Novel ctla4/cd28 ligands and uses therefor

ABSTRACT

Nucleic acids encoding novel CTLA4/CD28 ligands which costimulate T cell activation are disclosed. In one embodiment, the nucleic acid has a sequence which encodes a B lymphocyte antigen, B7-2. Preferably, the nucleic acid is a DNA molecule comprising at least a portion of a nucleotide sequence shown in FIG.  8,  SEQ ID NO:1 or FIG.  14,  SEQ ID NO:23. The nucleic acid sequences of the invention can be integrated into various expression vectors, which in turn direct the synthesis of the corresponding proteins or peptides in a variety of hosts, particularly eukaryotic cells, such as mammalian and insect cell culture. Also disclosed are host cells transformed to produce proteins or peptides encoded by the nucleic acid sequences of the invention and isolated proteins and peptides which comprise at least a portion of a novel B lymphocyte antigen. Proteins and peptides described herein can be administered to subjects to enhance or suppress T cell-mediated immune responses.

RELATED APPLICATIONS

[0001] This application is a continution-in-part of U.S. Ser. No.08/280,757, entitled “Novel CTLA4/CD28 Ligands and Uses Therefor” filedJul. 26, 1994, which is a continuation-in-part of U.S. Ser. No.08/109,393, entitled “Novel CTLA4/CD28 Ligands and Uses Therefor” filedAug. 19, 1993, which is a continuation-in-part of U.S. Ser. No.08/101,624, also entitled “Novel CTLA4/CD28 Ligands and Uses Therefor”,filed Jul. 26, 1993. This application is also a continution-in-part ofU.S. Ser. No. 08/147,773, entitled “Tumor Cells Modified To Express B7-2And B7-3 With Increased Inmmunogenicity And Uses Therefor” filed Nov. 3,1993. The contents of each of these applications is incorporated hereinby reference.

GOVERNMENT FUNDING

[0002] Work described herein was supported under CA-40216-08 awarded bythe National Institutes of Health. The U.S. government therefore mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] To induce antigen-specific T cell activation and clonalexpansion, two signals provided by antigen-presenting cells (APCs) mustbe delivered to the surface of resting T lymphocytes (Jenkins, M. andSchwartz, R. (1987) J. Exp. Med. 165, 302-319; Mueller, D. L., et al.(1990) J. Immunol 144, 3701-3709; Williams, I. R. and Unanue, E. R.(1990) J. Immunol. 145, 85-93). The first signal, which confersspecificity to the immune response, is mediated via the T cell receptor(TCR) following recognition of foreign antigenic peptide presented inthe context of the major histocompatibility complex (MHC). The secondsignal, termed costimulation, induces T cells to proliferate and becomefuinctional (Schwartz, R. H. (1990) Science 248, 1349-1356).Costimulation is neither antigen-specific, nor MHC restricted and isthought to be provided by one or more distinct cell surface moleculesexpressed by APCs (Jenkins, M. K., et al. (1988) J. Immunol. 140,3324-3330; Linsley, P. S., et al. (1991) J. Exp. Med. 173, 721-730;Gimmi, C. D., et al., (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579;Young, J. W., et al. (1992) J. Clin. Invest. 90, 229-237; Koulova, L.,et al. (1991) J. Exp. Med. 173, 759-762; Reiser, H., et al. (1992) Proc.Natl. Acad. Sci. USA. 89, 271-275; van-Seventer, G. A., et al. (1990) J.Immunol. 144, 4579-4586; LaSalle, J. M., et al., (1991) J. Immunol. 147,774-80; Dustin, M. I., et al., (1989) J. Exp. Med. 169, 503; Armitage,R. J., et al. (1992) Nature 357, 80-82; Liu, Y., et al. (1992) J. Exp.Med. 175, 437-445).

[0004] Considerable evidence suggests that the B7 protein, expressed onAPCs, is one such critical costimulatory molecule (Linsley, P. S., etal., (1991) J. Exp. Med. 173, 721-730; Gimmi, C. D., et al., (1991)Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Koulova, L., et al., (1991)J. Exp. Med. 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad.Sci. USA. 89, 271-275; Linsley, P. S. et al. (1990) Proc. Natl. Acad.Sci. USA. 87, 5031-5035; Freeman, G. J. et al. (1991) J. Exp. Med.174,625-631.). B7 is the counter-receptor for two ligands expressed on Tlymphocytes. The first ligand, termed CD28, is constitutively expressedon resting T cells and increases after activation. After signalingthrough the T cell receptor, ligation of CD28 induces T cells toproliferate and secrete IL-2 (Linsley, P. S., et al. (1991) J. Exp. Med.173, 721-730; Gimmi, C. D., et al. (1991) Proc. Natl. Acad Sci. USA. 88,6575-6579; Thompson, C. B., et al. (1989) Proc. Natl. Acad. Sci. USA.86, 1333-1337; June, C. H., et al. (1990) Immunol. Today. 11, 211-6;Harding, F. A., et al. (1992) Nature. 356, 607-609.). The second ligand,termed CTLA4 is homologous to CD28 but is not expressed on resting Tcells and appears following T cell activation (Brunet, J. F., et al.,(1987) Nature 328, 267-270). DNA sequences encoding the human and murineCTLA4 protein are described in Dariavich, et al. (1988) Eur. J. Immunol.18(12), 1901-1905; Brunet, J .F., et al. (1987) supra; Brunet, J. F. etal. (1988) Immunol. Rev. 103:21-36; and Freeman, G. J., et al. (1992) J.Immunol. 149, 3795-3801. Although B7 has a higher affinity for CTLA4than for CD28 (Linsley, P. S., et al., (1991) J. Exp. Med. 174,561-569), the function of CTLA4 is still unknown.

[0005] The importance of the B7:CD28/CTLA4 costimulatory pathway hasbeen demonstrated in vitro and in several in vivo model systems.Blockade of this costimulatory pathway results in the development ofantigen specific tolerance in murine and humans systems (Harding, F. A.,et al. (1992) Nature. 356, 607-609; Lenschow, D. J., et al. (1992)Science. 257, 789-792; Turka, L. A., et al. (1992) Proc. Natl. Acad.Sci. USA. 89, 11102-11105; Gimmi, C. D., et al. (1993) Proc. Natl. Acad.Sci USA 90, 6586-6590; Boussiotis, V., et al. (1993) J. Exp. Med. 178,1753-1763). Conversely, expression of B7 by B7 negative murine tumorcells induces T-cell mediated specific immunity accompanied by tumorrejection and long lasting protection to tumor challenge (Chen, L., etal. (1992) Cell 71, 1093-1102; Townsend, S. E. and Allison, J. P. (1993)Science 259, 368-370; Baskar, S., et al. (1993) Proc. Natl. Acad. Sci.90, 5687-5690.). Therefore, manipulation of the B7:CD28/CTLA4 pathwayoffers great potential to stimulate or suppress immune responses inhumans.

SUMMARY OF THE INVENTION

[0006] This invention pertains to isolated nucleic acids encoding novelmolecules which costimulate T cell activation. Preferred costimulatorymolecules include antigens on the surface of B lymphocytes, professionalantigen presenting cells (e.g., monocytes, dendritic cells, Langerhancells) and other cells (e.g., keratinocytes, endothelial cells,astrocytes, fibroblasts, oligodendrocytes) which present antigen toimmune cells, and which bind either CTLA4, CD28, both CTLA4 and CD28 orother known or as yet undefined receptors on immune cells. Suchcostimulatory molecules are referred to herein as CTLA4/CD28 bindingcounter-receptors or B lymphocyte antigens, and are capable of providingcostimulation to activated T cells to thereby induce T cellproliferation and/or cytokine secretion. Preferred B lymphocyte antigensinclude B7-2 and B7-3 and soluble fragments or derivatives thereof whichbind CTLA4 and/or CD28 and have the ability to inhibit or inducecostimulation of immune cells. In one embodiment, an isolated nucleicacid which encodes a peptide having the activity of the human B7-2 Blymphocyte antigen is provided. Preferably, the nucleic acid is a cDNAmolecule having a nucleotide sequence encoding human B7-2, as shown inFIG. 8 (SEQ ID NO:1). In another embodiment, the nucleic acid is a cDNAmolecule having a nucleotide sequence encoding murine B7-2, as shown inFIG. 14 (SEQ ID NO:22).

[0007] The invention also features nucleic acids which encode a peptidehaving B7-2 activity and at least about 50%, more preferably at leastabout 60% and most preferably at least about 70% homologous with anamino acid sequence shown in FIG. 8 (SEQ ID NO:2) or an amino acidsequence shown in FIG. 14 (SEQ ID NO:23). Nucleic acids which encodepeptides having B7-2 activity and at least about 80%, more preferably atleast about 90%, more preferably at least about 95% and most preferablyat least about 98% or at least about 99% homologous with an amino acidsequence shown in FIG. 8 (SEQ ID NO:2) or an amino acid sequence shownin FIG. 14 (SEQ ID NO:23) are also within the scope of the invention. Inanother embodiment, the peptide having B7-2 activity is encoded by anucleic acid which hybridizes under high or low stringency conditions toa nucleic acid which encodes a peptide having an amino acid sequence ofFIG. 8 (SEQ ID NO:2) or a peptide having an amino acid sequence shown inFIG. 14 (SEQ ID NO:23).

[0008] The invention further pertains to an isolated nucleic acidcomprising a nucleotide sequence encoding a peptide having B7-2 activityand having a length of at least 20 amino acid residues. Peptides havingB7-2 activity and consisting of at least 40 amino acid residues inlength, at least 60 amino acid residues in length, at least 80 aminoacid residues in length, at least 100 amino acid residues in length orat least 200 or more amino acid residues in length are also within thescope of this invention. Particularly preferred nucleic acids encode apeptide having B7-2 activity, a length of at least 20 amino acidresidues or more and at least 50% or greater homology (preferably atleast 70%) with a sequence shown in FIG. 8 (SEQ ID NO:2).

[0009] In one preferred embodiment, the invention features an isolatedDNA encoding a peptide having B7-2 activity and an amino acid sequencerepresented by a formula:

X_(n)—Y—Z_(m)

[0010] In the formula, Y consists essentially of amino acid residues24-245 of the sequence shown in FIG. 8 (SEQ ID NO:2). X_(n) and Z_(m)are additional amino acid residue(s) linked to Y by an amide bond. X_(n)and Z_(m) are amino acid residues selected from amino acid residuescontiguous to Y in the amino acid sequence shown in FIG. 8 (SEQ IDNO:2). X_(n) is amino acid residue(s) selected from amino acidscontiguous to the amino terminus of Y in the sequence shown in FIG. 8(SEQ ID NO:2), i.e., selected from amino acid residue 23 to 1. Z_(m) isamino acid residue(s) selected from amino acids contiguous to thecarboxy terminus of Y in the sequence shown in FIG. 8 (SEQ ID NO:2),i.e., selected from amino acid residue 246 to 329. According to theformula, n is a number from 0 to 23 (n=0-23) and m is a number from 0 to84 (m=0-84). A particularly preferred DNA encodes a peptide having anamino acid sequence represented by the formula X_(n)—Y—Z_(m), where Y isamino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO:2)and n=0 and m=0.

[0011] The invention also features an isolated DNA encoding a B7-2fusion protein which includes a nucleotide sequence encoding a firstpeptide having B7-2 activity and a nucleotide sequence encoding a secondpeptide corresponding to a moiety that alters the solubility, bindingaffinity, stability or valency of the first peptide. Preferably, thefirst peptide having B7-2 activity includes an extracellular domainportion of the B7-2 protein (e.g., about amino acid residues 24-245 ofthe sequence shown in FIG. 8 (SEQ ID NO:2)) and the second peptide is animmunoglobulin constant region, for example, a human Cγ1 or Cγ4 domain,including the hinge, CH2 and CH3 region, to produce a B7-2immunoglobulin fusion protein (B7-2Ig)(see Capon et al. (1989) Nature337, 525-531 and Capon U.S. Pat. No. 5,116,964).

[0012] The nucleic acids obtained in accordance with the presentinvention can be inserted into various expression vectors, which in turndirect the synthesis of the corresponding protein or peptides in avariety of hosts, particularly eucaryotic cells, such as mammalian andinsect cell culture, and procaryotic cells such as E. coli. Expressionvectors within the scope of the invention comprise a nucleic acidencoding at least one peptide having the activity of a novel Blymphocyte antigen as described herein, and a promoter operably linkedto the nucleic acid sequence. In one embodiment, the expression vectorcontains a DNA encoding a peptide having the activity of the B7-2antigen and a DNA encoding a peptide having the activity of another Blymphocyte antigen, such as the previously characterized B7 activationantigen, referred to herein as B7-1. Such expression vectors can be usedto transfect host cells to thereby produce proteins and peptides,including fusion proteins, encoded by nucleic acids as described herein.

[0013] Nucleic acid probes useful for assaying a biological sample forthe presence of B cells expressing the B lymphocyte antigens B7-2 andB7-3 are also within the scope of the invention.

[0014] The invention further pertains to isolated peptides having theactivity of a novel B lymphocyte antigen, including the B7-2 and B7-3protein antigens. A preferred peptide having B7-2 activity is producedby recombinant expression and comprises an amino acid sequence shown inFIG. 8 (SEQ ID NO: 2). Another preferred peptide having B7-2 activitycomprises an amino acid sequence shown in FIG. 14 (SEQ ID NO:23). Aparticularly preferred peptide having the activity of the B7-2 antigenincludes at least a portion of the mature form of the protein, such asan extracellular domain portion (e.g., about amino acid residues 24-245of SEQ ID NO:2) which can be used to enhance or suppress T-cell mediatedimmune responses in a subject. Other preferred peptides having B7-2activity include peptides having an amino acid sequence represented by aformula:

X_(n)—Y—Z_(m)

[0015] In the formula, Y is amino acid residues selected from the groupconsisting of: amino acid residues 55-68 of the sequence shown in FIG. 8(SEQ ID NO:2); amino acid residues 81-89 of the sequence shown in FIG. 8(SEQ ID NO:2); amino acid residues 128-142 of the sequence shown in FIG.8 (SEQ ID NO:2); amino acid residues 160-169 of the sequence shown inFIG. 8 (SEQ ID NO:2); amino acid residues 188-200 of the sequence shownin FIG. 8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequenceshown in FIG. 8 (SEQ ID NO:2). In the formula X_(n) and Z_(m) areadditional amino acid residue(s) linked to Y by an amide bond and areselected from amino acid residues contiguous to Y in the amino acidsequence shown in FIG. 8 (SEQ ID NO:2). X_(n) is amino acid residue(s)selected from amino acids contiguous to the amino terminus of Y in thesequence shown in FIG. 8 (SEQ ID NO:2). Z_(m) is amino acid residue(s)selected from amino acids contiguous to the carboxy terminus of Y in thesequence shown in FIG. 8 (SEQ ID NO:2). According to the formula, n is anumber from 0 to 30 (n=0-30) and m is a number from 0 to 30 (m=0-30).

[0016] Fusion proteins or hybrid fusion proteins including a peptidehaving the activity of a novel B lymphocyte antigen (e.g., B7-2, B7-3)are also featured. For example, a fusion protein comprising a firstpeptide which includes an extracellular domain portion of a novel Blymphocyte antigen fused to second peptide, such as an immunoglobulinconstant region, that alters the solubility, binding affinity, stabilityand/or valency of the first peptide are provided. In one embodiment, afusion protein is produced comprising a first peptide which includesamino acid residues of an extracellular domain portion of the B7-2protein joined to a second pepide which includes amino acid residues ofa sequence corresponding to the hinge, CH2 and CH3 regions of Cγ1 or Cγ4to form a B7-2Ig fusion protein. In another embodiment, a hybrid fusionprotein is produced comprising a first peptide which includes anextracellular domain portion of the B7-1 antigen and an extracellulardomain portion of the B7-2 antigen and a second peptide which includesamino acid residues corresponding to the hinge, CH2 and CH3 of Cγ1 (seee.g., Linsley et al. (1991) J. Exp. Med. 1783:721-730; Capon et al.(1989) Nature 33, 525-531; and Capon U.S. Pat. No. 5,116,964). In a yetanother embodiment, a hybrid fusion protein comprises theimmuoglobulin-like variable domain of B7-2, but not theimmunoglobulin-like constant domain of B7-2, linked to the constantregion of an immunoglobulin molecule. In a preferred embodiment, theB7-2Ig fusion protein includes the variable domain of human B7-2,preferably from about amino acid residue 24 to about amino acid residue133 of the human B7-2 protein (as shown SEQ ID NO: 2), fused to theconstant region of an IgG molecule.

[0017] Isolated peptides and fusion proteins of the invention can beadministered to a subject to either upregulate or inhibit the expressionof one or more B lymphocyte antigens or block the ligation of one ormore B lymphocyte antigens to their natural ligand on immune cells, suchas T cells, to thereby provide enhancement or suppression ofcell-mediated immune responses in vivo.

[0018] Another embodiment of the invention provides antibodies,preferably monoclonal antibodies, specifically reactive with a peptideof a novel B lymphocyte antigen or fusion protein as described herein.Preferred antibodies are anti-human B7-2 monoclonal antibodies producedby hybridoma cells HF2.3D1, HA5.2B7 and HA3.1F9. These hybridoma cellshave been deposited with the American Type Culture Collection at ATCCAccession No. (HF2.3D1), ATCC Accession No. (HA5.2B7), and ATCCAccession No. (HA3.1F9).

[0019] A still further aspect of the invention involves the use of thenucleic acids of the invention, especially the cDNAs, to enhance theimmunogenicity of a mammalian cell. In preferred embodiments, themammalian cell is a tumor cell, such as a sarcoma, a lymphoma, amelanoma, a neuroblastoma, a leukemia or a carcinoma, or an antigenpresenting cell, such as a macrophage, which is transfected to allowexpression of a peptide having the activity of a novel B lymphocyteantigen of the invention on the surface of the cell. Macrophages thatexpress a peptide having the activity of a B lymphocyte antigen, such asthe B7-2 antigen, can be used as antigen presenting cells, which, whenpulsed with an appropriate pathogen-related antigen or tumor antigen,enhance T cell activation and immune stimulation.

[0020] Mammalian cells can be transfected with a suitable expressionvector containing a nucleic acid encoding a peptide having the activityof a novel B lymphocyte antigen, such as the B7-2 antigen, ex vivo andthen introduced into the host mammal, or alternatively, cells can betransfected with the gene in vivo via gene therapy techniques. Forexample, the nucleic acid encoding a peptide having B7-2 activity can betransfected alone, or in combination with nucleic acids encoding othercostimulatory molecules. In enhancing the immunogenicity of tumors whichdo not express Class I or Class II MHC molecules, it may be beneficialto additionally transfect appropriate class I or II genes into themammalian cells to be transfected with a nucleic acid encoding a peptidehaving the activity of a B lymphocyte antigen, as described herein.

[0021] The invention also provides methods for inducing both generalimmunosuppression and antigen-specific tolerance in a subject by, forexample, blocking the functional interaction of the novel B lymphocyteantigens of the invention, e.g., B7-2 and B7-3, to their naturalligand(s) on T cells or other immune system cells, to thereby blockco-stimulation through the receptor-ligand pair. In one embodiment,inhibitory molecules that can be used to block the interaction of thenatural human B7-2 antigen to its natural ligands (e.g., CTLA4 and CD28)include a soluble peptide having B7-2 binding activity but lacking theability to costimulate immune cells, antibodies that block the bindingof B7-2 to its ligands and fail to deliver a co-stimulatory signal (socalled “blocking antibodies”, such as blocking anti-B7-2 antibodies),B7-2-Ig fusion proteins, which can be produced in accordance with theteachings of the present invention, as well as soluble forms of B7-2receptors, such as CTLA4Ig or CD28Ig. Such blocking agents can be usedalone or in combination with agents which block interaction of othercostimulatory molecules with their natural ligands (e.g., anti-B7antibody). Inhibition of T cell responses and induction of T celltolerance according to the methods described herein may be usefulprophylactically, in preventing transplantation rejection (solid organ,skin and bone marrow) and graft versus host disease, especially inallogeneic bone marrow transplantation. The methods of the invention mayalso be useful therapeutically, in the treatment of autoimmune diseases,allergy and allergic reactions, transplantation rejection, andestablished graft versus host disease in a subject.

[0022] Another aspect of the invention features methods for upregulatingimmune responses by delivery of a costimulatory signal to T cellsthrough use of a stimulatory form of B7-2 antigen, which includesoluble, multivalent forms of B7-2 protein, such as a peptide havingB7-2 activity and B7-2 fusion proteins. Delivery of a stimulatory formof B7-2 in conjunction with antigen may be useful prophylactically toenhance the efficacy of vaccination against a variety of pathogens andmay also be useful therapeutically to upregulate an immune responseagainst a particular pathogen during an infection or against a tumor ina tumor-bearing host.

[0023] The invention also features methods of identifying moleculeswhich can inhibit either the interaction of B lymphocyte antigens, e.g.,B7-2, B7-3, with their receptors or interfere with intracellularsignalling through their receptors. Methods for identifying moleculeswhich can modulate the expression of B lymphocyte antigens on cells arealso provided. In addition, methods for identifying cytokines producedin response to costimulation of T cells by novel B lymphocyte antigensare within the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-B are graphic representations of the responses of CD28⁺T cells, as assessed by ³H-thymidine incorporation or IL-2 secretion, tocostimulation provided by either B7 (B7-1) transfected CHO cells (panela) or syngeneic activated B lymphocytes (panel b) cultured in media,anti-CD3 alone, or anti-CD3 in the presence of the following monoclonalantibodies or recombinant proteins: αB7 (133, anti-B7-1); CTLA4Ig; Fab αCD28; control Ig fusion protein (isotype control for CTLA4Ig); or αB5(anti-B5, the isotype control for anti-B7-1).

[0025] FIGS. 2A-C are graphs of log fluorescence intensity of cellsurface expression of B7-1 on splenic B cells activated with surfaceimmunoglobulin (sIg) crosslinking. The total (panel a), B7-1 positive(B7-1⁺, panel b) and B7-1 negative (B7-1⁻, panel c) activated B cel werestained with anti-B7-1 monoclonal antibody (133) and fluorosceinisothiocyanate (FITC) labeled goat anti-mouse immunoglobulin andanalyzed by flow cytometry.

[0026] FIGS. 3A-B are graphic representations of the responses of CD28⁺T cells, as assessed by ³H-thymidine incorporation and IL-2 secretion,to costimulation provided by B7-1⁺ (panel a) or B7-1⁻ (panel b)activated syngeneic B lymphocytes cultured in media, anti-CD3 alone, oranti-CD3 in the presence of the following monoclonal antibodies orrecombinant proteins: αBB-1 (133, anti-B7-1 and anti-B7-3); αB7(anti-B7-1); CTLA4Ig; Fab αCD28; control Ig fusion protein or αB5(anti-B5).

[0027]FIG. 4 is a graphic representation of the cell surface expressionof B7-1, B7-3 and total CTLA4 counter-receptors on fractionated B7-1⁺and B7-1⁻ activated B lymphocytes.

[0028]FIG. 5 is a graphic representation of temporal surface expressionof B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BB1) andB7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by sIgcrosslinking.

[0029]FIG. 6 is a graphic representation of temporal surface expressionof B7-1 (CTLA4Ig and mAbs BB-1 and 133), B7-3 (CTLA4Ig and mAb BB1) andB7-2 (CTLA4Ig) counter-receptors on splenic B cells activated by MHCclass II crosslinking.

[0030] FIGS. 7A-B are graphic representations of the response of CD28⁺ Tcells, as assessed by ³H-thymidine incorporation and IL-2 secretion, tocostimulation provided by syngeneic B lymphocytes activated by sIgcrosslinking for 24 hours (panel a) or 48 hours (panel b) and culturedin media, anti-CD3 alone, or anti-CD3 in the presence of the followingmonoclonal antibodies or recombinant protein: αB7(133, anti-B7-1); αBB1(anti-B7-1, anti-B7-3) CTLA4Ig; Fab αCD28; and αB5(anti-B5).

[0031]FIG. 8 is the nucleotide and deduced amino acid sequence of thehuman B lymphocyte antigen B7-2 (hB7-2-clone29).

[0032]FIG. 9 is a graphic representation of COS cells transfected withcontrol plasmid (pCDNAI), plasmid expressing B7-1 (B7-1), or plasmidexpressing B7-2 (B7-2) stained with either control mAb (IgM), anti-B7-1(mAbs 133 and BB-1), recombinant protein CTLA4Ig, or isotype matchedcontrol Ig protein followed by the appropriate second FITC labelledimmunoglobulin and analyzed by flow cytometry.

[0033] FIGS. 10A-B show RNA blot analyses of B7-2 expression inunstimulated and anti-Ig activated human spenic B cells and cell lines(panel a) and human myelomas (panel b).

[0034]FIG. 11 is a graphic representation of the proliferation of CD28+T cells, as assessed by ³H-thymidine incorporation or IL-2 secretion, tosubmitogenic stimulation with phorbol myristic acid (PMA) and COS cellstransfected with vector alone or vectors directing the expression ofeither B7-1 or B7-2.

[0035]FIG. 12 is a graphic representation of the inhibition by mAbs andrecombinant proteins of the proliferation of CD28+ T cells, as assessedby ³H-thymidine incorporation and IL-2 secretion, to stimulation by PMAand COS cells transfected with vector alone (vector), or with a vectorexpressing B7-1 (B7-1) or B7-2 (B7-2). Inhibition studies were performedwith the addition of either no antibody (no mAb), anti-B7 mAb 133 (133),anti-B7 mAb BB-1 (BB1), anti-B5 mAb (B5), Fab fragment of anti-CD28(CD28 Fab), CTLA4Ig (CTLA4Ig), or Ig control protein (control Ig) to thePMA stimulated COS cell admixed CD28⁺ T cells.

[0036]FIG. 13 shows the sequence homology between the human B7-2 protein(h B7-2) deduced amino acid sequence (SEQ ID NO: 2) and the amino acidsequence of both the human B7-1 protein (h B7-1) (SEQ ID NO: 28 and 29)and the murine B7-1 protein (m B7) (SEQ ID NO: 30 and 31).

[0037]FIG. 14 is the nucleotide and deduced amino acid sequence of themurine B7-2 antigen (mB7-2) (SEQ ID NO: 22 and 23).

[0038]FIG. 15 is a graphic representation of the competitive inhibitionof binding of biotinylated-CTLA4Ig to immobilized B7-2 Ig by B7family-Ig fusion proteins. The Ig fusion proteins examined ascompetitors were: full-length B7-2 (hB7.2), full-length B7-1 (hB7.1),the variable region-like domain of B7-2 (hB7.2V) or the constantregion-like domain of B7-2 (hB7.2C).

[0039] FIGS. 16A-B are graphic representations of the competitiveinhibition of binding of biotinylated-B7-1-Ig (panel A) or B7-2-Ig(panel B) to immobilized CTLA4-Ig by increasing concentrations ofunlabelled B7-1-Ig (panel A) or B7-2-Ig (panel B). The experimentallydetermined IC₅₀ values are indicated in the upper right comer of thepanels.

[0040]FIG. 17 depicts flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HA3.1F9. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

[0041]FIG. 18 depicts flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HA5.2B7. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

[0042]FIG. 19 depicts flow cytometric profiles of cells stained with ananti-hB7-2 monoclonal antibody, HF2.3D1. Cells stained with the antibodywere CHO cells transfected to express human B7-2 (CHO-hB7.2), NIH 3T3cells transfected to express human B7-2 (3T3-hB7.2) and controltransfected NIH 3T3 cells (3T3-neo). The anti-hB7.2 antibody B70 wasused as a positive control.

[0043]FIG. 20 is a graphic representation of the direct binding ofsoluble biotinylated CTLA4Ig to B7-1Ig, B7-1VIg, B7-1CIg, B7-2Ig,B7-2VIg, B7-2CIg, or human IgG (hIgG) bound to plates.

[0044] FIGS. 21A-E depict flow cytometric profiles of binding of B7-2Ig(Panel C), B7-2VIg (Panel D), B7-1Ig (Panel E), or secondary antibodyalone (Panel B) to CTLA4+ CHO cells. Panel A is a negative controlrepresenting untransfected CHO cells.

[0045]FIG. 22 depicts flow cytometric profiles of binding of control Ig,B7-1Ig, B7-2Ig, B7-2VIg, and anti-CD28 to CHO cells expressing CD28.

[0046]FIG. 23 represents a histogram showing proliferation of CD28+ Tcells stimulated with 1 ng/ml PMA alone or with either of the followingcostimulatory signals: CHO/B7-1 cells, CHO/B7-2 cells, control Ig (30μg/ml), or B7-1Ig, B7-2Ig, or B7-2VIg (30 μg or 100 μg/ml each).

[0047]FIG. 24 represents a histogram showing proliferation of, and IL-2production by CD28+ T cells incubated with anti-CD3 attached to platesand B7-1Ig (10, 3 or 1 μg/ml) or B7-2Ig (19, 3 or 1 μg/ml) or B7-2VIg(3.0-0.01 μg/ml).

[0048]FIG. 25 represents the amount of IL-2 produced by CD28+ T cellsafter 1, 2, or 3 days of incubation of the cells with anti-CD3 alone ortogether with either CHO/B7-2 cells or B7-2VIg fusion protein.

[0049]FIG. 26 represents the amount of IL-2 secreted by CD28+ T cellsafter 1, 2, or 5 days of incubation of the cells with anti-CD3 alone orwith either anti-CD28, B7-1Ig, B7-2Ig, or B7-2VIg.

[0050]FIG. 27 is a graphical representation of the growth of CD28+ Tcells incubated with anti-CD3 alone, or with B7-1Ig, B7-2Ig, B7-2VIg, ortogether with either anti-CD28.

DETAILED DESCRIPTION OF THE INVENTION

[0051] In addition to the previously characterized B lymphocyteactivation antigen B7 (referred to herein as B7-1), human B lymphocytesexpress other novel molecules which costimulate T cell activation. Thesecostimulatory molecules include antigens on the surface of Blymphocytes, professional antigen presenting cells (e.g., monocytes,dendritic cells, Langerhan cells) and other cells (e.g., keratinocytes,endothelial cells, astrocytes, fibroblasts, oligodendrocytes) whichpresent antigen to immune cells, and which bind either CTLA4, CD28, bothCTLA4 and CD28 or other known or as yet undefined receptors on immunecells. Costimulatory molecules within the scope of the invention arereferred to herein as CTLA4/CD28 ligands (counter-receptors) or Blymphocyte antigens. Novel B lymphocyte antigens which providecotimulation to activated T cells to thereby induce T cell proliferationand/or cytokine secretion include the B7-2 (human and murine) and theB7-3 antigens described and characterized herein.

[0052] The B lymphocyte antigen B7-2 is expressed by human B cells atabout 24 hours following stimulation with either anti-immunoglobulin oranti-MHC class II monoclonal antibody. The B7-2 antigen inducesdetectable IL-2 secretion and T cell proliferation. At about 48 to 72hours post activation, human B cells express both B7-1 and a third CTLA4counter-receptor, B7-3, identified by a monoclonal antibody BB-1, whichalso binds B7-1 (Yokochi, T., et al. (1982) J. Immunol. 128, 823-827).The B7-3 antigen is also expressed on B7-1 negative activated B cellsand can costimulate T cell proliferation without detectable IL-2production, indicating that the B7-1 and B7-3 molecules are distinct.B7-3 is expressed on a wide variety of cells including activated Bcells, activated monocytes, dendritic cells, Langerhan cells andkeratinocytes. At 72 hours post B cell activation, the expression ofB7-1 and B7-3 begins to decline. The presence of these costimulatorymolecules on the surface of activated B lymphocytes indicates that Tcell costimulation is regulated, in part, by the temporal expression ofthese molecules following B cell activation.

[0053] Accordingly, one aspect of this invention pertains to isolatednucleic acids comprising a nucleotide sequence encoding a novelcostimulatory molecule, such as the B lymphocyte antigen, B7-2,fragments of such nucleic acids, or equivalents thereof. The term“nucleic acid” as used herein is intended to include such fragments orequivalents. The term “equivalent” is intended to include nucleotidesequences encoding functionally equivalent B lymphocyte antigens orfunctionally equivalent peptides having an activity of a novel Blymphocyte antigen, i.e., the ability to bind to the natural ligand(s)of the B lymphocyte antigen on immune cells, such as CTLA4 and/or CD28on T cells, and inhibit (e.g., block) or stimulate (e.g., enhance)immune cell costimulation. Such nucleic acids are considered equivalentsof the human B7-2 nucleotide sequence provided in FIG. 8 (SEQ ID NO:1)and the murine B7-2 nucleotide sequence provided in FIG. 14 (SEQ IDNO:22) and are within the scope of this invention.

[0054] In one embodiment, the nucleic acid is a cDNA encoding a peptidehaving an activity of the B7-2 B lymphocyte antigen. Preferably, thenucleic acid is a cDNA molecule consisting of at least a portion of anucleotide sequence encoding human B7-2, as shown in FIG. 8 (SEQ IDNO:1) or at least a portion of a nucleotide sequence encoding murineB7-2, as shown in FIG. 14 (SEQ ID NO:22). A preferred portion of thecDNA molecule of FIG. 8 (SEQ ID NO:1) or FIG. 14 (SEQ ID NO:22) includesthe coding region of the molecule.

[0055] In another embodiment, the nucleic acid of the invention encodesa peptide having an activity of B7-2 and comprising an amino acidsequence shown in FIG. 8 (SEQ ID NO:2) or FIG. 14 (SEQ ID NO:23).Preferred nucleic acids encode a peptide having B7-2 activity and atleast about 50% homology, more preferably at least about 60% homologyand most preferably at least about 70% homology with an amino acidsequence shown in FIG. 8 (SEQ ID NO:2). Nucleic acids which encodepeptides having B7-2 activity and at least about 90%, more preferably atleast about 95%, and most preferably at least about 98-99% homologouswith a sequence set forth in FIG. 8 (SEQ ID NO:2) are also within thescope of the invention. Homology refers to sequence similarity betweentwo peptides having the activity of a novel B lymphocyte antigen, suchas B7-2, or between two nucleic acid molecules. Homology can bedetermined by comparing a position in each sequence which may be alignedfor purposes of comparison. When a position in the compared sequences isoccupied by the same nucleotide base or amino acid, then the moleculesare homologous at that position. A degree (or percentage) of homologybetween sequences is a function of the number of matching or homologouspositions shared by the sequences.

[0056] Another aspect of the invention provides a nucleic acid whichhybridizes under high or low stringency conditions to a nucleic acidwhich encodes a peptide having all or a portion of an amino acidsequence shown in FIG. 8 (SEQ ID NO:2) or a peptide having all or aportion of an amino acid sequence shown in FIG. 14 (SEQ ID NO:23).Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C. are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C. to highstringency conditions, at about 65° C.

[0057] Isolated nucleic acids encoding a peptide having an activity of anovel B lymphocyte antigen, as described herein, and having a sequencewhich differs from nucleotide sequence shown in FIG. 8 (SEQ ID NO:1) orFIG. 14 (SEQ ID NO:22) due to degeneracy in the genetic code are alsowithin the scope of the invention. Such nucleic acids encodefunctionally equivalent peptides (e.g., a peptide having B7-2 activity)but differ in sequence from the sequence of FIG. 8 or FIG. 14 due todegeneracy in the genetic code. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may occur due to degeneracy in the genetic code. As one example, DNAsequence polymorphisms within the nucleotide sequence of a B7-2(especially those within the third base of a codon) may result in“silent” mutations in the DNA which do not affect the amino acidencoded. However, it is expected that DNA sequence polymorphisms that dolead to changes in the amino acid sequences of the B7-2 antigen willexist within a population. It will be appreciated by one skilled in theart that these variations in one or more nucleotides (up to about 3-4%of the nucleotides) of the nucleic acids encoding peptides having theactivity of a novel B lymphocyte antigen may exist among individualswithin a population due to natural allelic variation. Any and all suchnucleotide variations and resulting amino acid polymorphisms are withinthe scope of the invention. Furthermore, there may be one or moreisoforms or related, cross-reacting family members of the novel Blymphocyte antigens described herein. Such isoforms or family membersare defined as proteins related in function and amino acid sequence to aB lymphocyte antigen (e.g., the B7-2 antigen), but encoded by genes atdifferent loci.

[0058] A “fragment” of a nucleic acid encoding a novel B lymphocyteantigen is defined as a nucleotide sequence having fewer nucleotidesthan the nucleotide sequence encoding the entire amino acid sequence ofthe B lymphocyte antigen and which encodes a peptide having an activityof the B lymphocyte antigen (i.e., the ability to bind to the naturalligand(s) of the B lymphocyte antigen on immune cells, such as CTLA4and/or CD28 on T cells and either stimulate or inhibit immune cellcostimulation). Thus, a peptide having B7-2 activity binds CTLA4 and/orCD28 and stimulates or inhibits a T cell mediated immune response, asevidenced by, for example, cytokine production and/or T cellproliferation by T cells that have received a primary activation signal.In one embodiment, the nucleic acid fragment encodes a peptide of theB7-2-antigen which retains the ability of the antigen to bind CTLA4and/or CD28 and deliver a costimulatory signal to T lymphocytes. Inanother embodiment, the nucleic acid fragment encodes a peptideincluding an extracellular portion of the human B7-2 antigen (e.g.,approximately amino acid residues 24-245 of the sequence provided inFIG. 8 (SEQ ID NO:2)) which can be used to bind CTLA4 and/or CD28 and,in monovalent form, inhibit costimulation, or in multivalent form,induce or enhance costimulation.

[0059] Preferred nucleic acid fragments encode peptides of at least 20amino acid residues in length, preferably at least 40 amino acidresidues and length, and more preferably at least 60 amino acid residuesin length. Nucleic acid fragments which encode peptides of at least 80amino acid residues in length, at least 100 amino acid residues inlength, and at least 200 or more amino acids in length are also withinthe scope of the invention. Particularly preferred nucleic acidfragments encode a peptide having the activity of human B7-2 and anamino acid sequence represented by a formula:

X_(n)—Y—Z_(m)

[0060] In the fomula, Y comprises amino acid residues 24-245 of thesequence shown in FIG. 8 (SEQ ID NO:2). X_(n) and Z_(m) are additionalamino acid residue(s) linked to Y by an amide bond. X_(n) and Z_(m) areselected from amino acid residues contiguous to Y in the amino acidsequence shown in FIG. 8 (SEQ ID NO:2). In the formula, X_(n) is aminoacid residue(s) selected from amino acids contiguous to the aminoterminus of Y in the sequence shown in FIG. 8 (SEQ ID NO:2), i.e., fromamino acid residue 23 to 1. Z_(m) is amino acid residue(s) selected fromamino acids contiguous to the carboxy terminus of Y in the sequenceshown in FIG. 8 (SEQ ID NO:2), i.e., from amino acid residue 246 to 329.In addition, in the formula, n is a number from 0 to 23 (n=0-23) and mis a number from 0 to 84 (m=0-84). A particularly preferred peptide hasan amino acid sequence represented by the formula X_(n)—Y—Z_(m) asabove, where n=0 and m=0.

[0061] Nucleic acid fragments within the scope of the invention includethose capable of hybridizing with nucleic acid from other animal speciesfor use in screening protocols to detect novel proteins that arecross-reactive with the B lymphocyte antigens described herein. Theseand other fragments are described in detail herein. Generally, thenucleic acid encoding a fragment of a B lymphocyte antigen will beselected from the bases coding for the mature protein, however, in someinstances it may be desirable to select all or part of a fragment orfragments from the leader sequence or non-coding portion of a nucleotidesequence. Nucleic acids within the scope of the invention may alsocontain linker sequences, modified restriction endonuclease sites andother sequences useful for molecular cloning, expression or purificationof recombinant protein or fragments thereof. These and othermodifications of nucleic acid sequences are described in further detailherein.

[0062] A nucleic acid encoding a peptide having an activity of a novel Blymphocyte antigen, such as the B7-2 antigen, may be obtained from mRNApresent in activated B lymphocytes. It should also be possible to obtainnucleic acid sequences encoding B lymphocyte antigens from B cellgenomic DNA. For example, the gene encoding the B7-2 antigen can becloned from either a cDNA or a genomic library in accordance withprotocols herein described. A cDNA encoding the B7-2 antigen can beobtained by isolating total mRNA from an appropriate cell line. Doublestranded cDNAs can then prepared from the total mRNA. Subsequently, thecDNAs can be inserted into a suitable plasmid or viral (e.g.,bacteriophage) vector using any one of a number of known techniques.Genes encoding novel B lymphocyte antigens can also be cloned usingestablished polymerase chain reaction techniques in accordance with thenucleotide sequence information provided by the invention. The nucleicacids of the invention can be DNA or RNA. A preferred nucleic acid is acDNA encoding the human B7-2 antigen having the sequence depicted inFIG. 8 (SEQ ID NO:1). Another preferred nucleic acid is a cDNA encodingthe murine B7-2 antigen having the sequence shown on FIG. 14 (SEQ IDNO:22).

[0063] This invention further pertains to expression vectors containinga nucleic acid encoding at least one peptide having the activity of anovel B lymphocyte antigen, as described herein, operably linked to atleast one regulatory sequence. “Operably linked” is intended to meanthat the nucleotide acid sequence is linked to a regulatory sequence ina manner which allows expression of the nucleotide sequence (e.g., incis or trans). Regulatory sequences are art-recognized and are selectedto direct expression of the desired protein in an appropriate host cell.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Such regulatory sequences areknown to those skilled in the art or one described in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transfected and/or the type of protein desired to beexpressed. In one embodiment, the expression vector includes a nucleicacid encoding at least a portion of the B7-2 protein, such as anextracellular domain portion. In another embodiment, the expressionvector includes a DNA encoding a peptide having an activity of the B7-2antigen and a DNA encoding a peptide having an activity of another Blymphocyte antigen, such as B7-1. cDNAs encoding the human B7-1 andmouse B7-1 antigens are shown in SEQ ID NO:28 and SEQ ID NO:30,respectively. The deduced amino acid sequences of these antigens arealso shown in SEQ ID NO:29 and SEQ ID NO:31, respectively. Suchexpression vectors can be used to transfect cells to thereby produceproteins or peptides, including fusion proteins or peptides encoded bynucleic acid sequences as described herein. These and other embodimentsare described in further detail herein.

[0064] The invention also features methods of producing peptides havingan activity of a novel B lymphocyte antigen. For example, a host celltransfected with a nucleic acid vector directing expression of anucleotide sequence encoding a peptide having an activity of the B7-2protein can be cultured in a medium under appropriate conditions toallow expression of the peptide to occur. In addition, one or moreexpression vectors containing DNA encoding a peptide having an activityof B7-2 and DNA encoding another peptide, such as a peptide having anactivity of a second B lymphocyte antigen (e.g., B7-1, B7-3) can be usedto transfect a host cell to coexpress these peptides or produce fusionproteins or peptides. In one embodiment, a recombinant expression vectorcontaining DNA encoding a B7-2 fusion protein is produced. A B7-2 fusionprotein can be produced by recombinant expression of a nucleotidesequence encoding a first peptide having B7-2 activity and a nucleotidesequence encoding second peptide corresponding to a moiety that altersthe solubility, affinity, stability or valency of the first peptide, forexample, an immunoglobulin constant region. Preferably, the firstpeptide consists of a portion of the extracellular domain of the humanB7-2 antigen (e.g., approximately amino acid residues 24-245 of thesequence shown in FIG. 8 (SEQ ID NO:2)). The second peptide can includean immunoglobulin constant region, for example, a human Cγ1 domain orCγ4 domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, orhuman IgCγ4, see e.g., Capon et al. U.S. Pat. No. 5,116,964,incorporated herein by reference). A resulting B7-2Ig fusion protein mayhave altered B7-2 solubility, binding affinity, stability and/or valency(i.e., the number of binding sites available per molecule) and mayincrease the efficiency of protein purification. Fusion proteins andpeptides produced by recombinant technique may be secreted and isolatedfrom a mixture of cells and medium containing the protein or peptide.Alternatively, the protein or peptide may be retained cytoplasmicallyand the cells harvested, lysed and the protein isolated. A cell culturetypically includes host cells, media and other byproducts. Suitablemediums for cell culture are well known in the art. Protein and peptidescan be isolated from cell culture medium, host cells, or both usingtechniques known in the art for purifying proteins and peptides.Techniques for transfecting host cells and purifying proteins andpeptides are described in further detail herein.

[0065] Particularly preferred human B7-2Ig fusion proteins include theextracellular domain portion or variable region-like domain of humanB7-2 coupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications which reduce oreliminate effector activity inherent in the immunoglobulin structure.For example, DNA encoding the extracellular portion of human B7-2(hB7-2), as well as DNA encoding the variable region-like domain ofhuman B7-2 (hB7.2V) or the constant region-like domain of human B7-2(hB7.2C) can be joined to DNA encoding the hinge, CH2 and CH3 regions ofhuman IgCγ1 and/or IgCγ4 modified by site directed mutagenesis. Thepreparation and characterization of these fusion proteins is describedin detail in Example 7.

[0066] In a specific embodiment, the protein of the invention is avariable region form of the B cell activation antigen B7-2. The language“a variable region form of the B cell activation antigen B7-2” isintended to include forms of B7-2 which contain the immunoglobulin-likevariable domain of B7-2, but do not comprise the immunoglobulin-likeconstant domain of B7-2. In a preferred embodiment, the variable regionform of B7-2 comprises an amino acid sequence starting at about aminoacid postion 18 to 30 and ending about amino acid position 128 to 140 ofhuman B7-2 protein (SEQ ID NO: 2). In a most preferred embodiment, thevariable form of B7-2 comprises about amino acids 24 to 133 of humanB7-2 protein (SEQ ID NO: 2). The variable region form of B7-2 canfurther be operatively linked directly to a transmembrane domain, suchas the transmembrane domain of B7-2, to form a variable region form ofB7-2 that can be expressed on a cell surface. “Operatively” is intendedto mean in such a way that the molecule formed by operatively linkingtwo or more domains or peptides is functional. The transmembrane domainof human B7-2 comprises about amino residues 246 to 268 of human B7-2protein. Thus, in one embodiment, the variable region form of B7-2 isoperatively linked to a peptide having a first amino acid locatedbetween about amino acid residue 238 and about amino acid residue 252,and a last amino acid residue located between about amino acid residue260 and about amino acid residue 274 of human B7-2 protein of sequenceSEQ ID NO: 2. The incorporation of a transmembrane domain in a proteinof the invention, allows the protein to be expressed on a cell surfacewhen a nucleic acid encoding the protein is expressed in the cell.

[0067] In another embodiment, the variable region form of B7-2 soperatively linked to a cytoplasmic domain, such as a cytoplasmic domainof B7-2. The cytoplasmic domain of human B7-2 comprises about amino acidresidues 269 to 329 of human B7-2 protein of SEQ ID NO: 2. Accordingly,in one embodiment, the variable region form of B7-2 is operativelylinked to a second B7-2 peptide, having a first amino acid residuelocated between about amino acids 260 and 275 of human B7-2 and aterminal amino acid residue located between about amino acid 323 toabout amino acid 335 of human B7-2 of SEQ ID NO: 2. In anotherembodiment, a variable region form of B7-2 is operatively linked to asecond B7-2 peptide of about amino acid residues 269 to 329 of humanB7-2.

[0068] In a further embodiment, a variable region form of B7-2operatively linked to a peptide corresponding to about the transmembranedomain of B7-2 is further operatively linked to a peptide correspondingsignificantly to the cytoplasmic domain of B7-2. Thus, proteins withinthe scope of the invention include those comprising an amino acidsequence from about position 24 to about position 133 of SEQ ID NO: 2,operatively linked to an amino acid sequence from about position 246 toabout position 268 of SEQ ID NO: 2 (V-region and transmembrane domains).Other proteins within the scope of the invention include thosecomprising an amino acid sequence from about position 24 to aboutposition 133 of SEQ ID NO: 2, operatively linked to an amino acidsequence from about position 246 to about position 329 of SEQ ID NO: 2(V-region, transmembrane and cytoplasmic domains). Yet other proteinswithin the scope of the invention include the leader sequence of B7-2(e.g. positions 1-23) at the N-terminus. Proteins including otherportions of B7-2 protein operatively linked to each other, but notincluding the immunoglobulin-like constant domain of B7-2, are alsowithin the scope of the invention. In other embodiments, B7-2 proteinsthat contain an immunoglobulin-like constant domain of B7-2 in theabsence of the variable region are contemplated.

[0069] A variable region form of B7-2, can also be linked to at leastone heterologous polypeptide. The term “heterologous polypeptide” isintended to include any polypeptide, such as a polypeptide that directsthe protein of the invention to a specific cellular compartment. In oneembodiment, the heterologous polypeptide is a signal peptide that allowsfor the protein to be secreted from the cell. Another heterologouspolypeptide within the scope of the invention is a signal peptide thatallows for the protein to be expressed on the surface of the cell. Inyet another embodiment, the heterologous polypeptide is a constantregion of an immunoglobulin molecule. In an even more preferredembodiment, the heterologous polypeptide comprises the hinge, CH2, andCH3 domains of IgG1, as described herein.

[0070] The variable region form of B7-2 can further be attached to alinker polypeptide. A “linker polypeptide” as defined herein includesany polypeptide that bridges two peptides in the protein of theinvention. Alternatively, the linker peptide is attached to either orboth ends of the protein. Thus, a linker peptide attached to one or bothends of the protein can for example facilitate binding of the protein ofthe invention to a solid support. The linker peptide can also be afragment of a bacterial or viral protein.

[0071] The fusion proteins described above can be, for example, human ormurine. The nucleic acid molecules encoding the above described fusionproteins, as well as expression vectors and host cells expressing thefusion proteins are also within the scope of the invention.

[0072] Transfected cells which express peptides having an activity ofone or more B lymphocyte antigens (e.g., B7-2, B7-3) on the surface ofthe cell are also within the scope of this invention. In one embodiment,a host cell such as a COS cell is transfected with an expression vectordirecting the expression of a peptide having B7-2 activity on thesurface of the cell. Such a transfected host cell can be used in methodsof identifying molecules which inhibit binding of B7-2 to itscounter-receptor on T cells or which interfere with intracellularsignaling of costimulation to T cells in response to B7-2 interaction.In another embodiment, a tumor cell such as a sarcoma, a melanoma, aleukemia, a lymphoma, a carcinoma or a neuroblastoma is transfected withan expression vector directing the expression of at least one peptidehaving the activity of a novel B lymphocyte antigen on the surface ofthe tumor cell. In some instances, it may be beneficial to transfect atumor cell to coexpress major histocompatibility complex (MHC) proteins,for example MHC class II α and β chain proteins or an MHC class I αchain protein, and, if necessary, a β2 microglobulin protein. Suchtransfected tumor cells can be used to induce tumor immunity in asubject. These and other embodiments are described in further detailherein.

[0073] The nucleic acid sequences of the invention can also bechemically synthesized using standard techniques. Various methods ofchemically synthesizing polydeoxynucleotides are known, includingsolid-phase synthesis which, like peptide synthesis, has been fullyautomated in commercially available DNA synthesizers (See e.g., Itakuraet al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No.4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,incorporated by reference herein).

[0074] Another aspect of the invention pertains to isolated peptideshaving an activity of a novel B lymphocyte antigen (e.g., B7-2, B7-3). Apeptide having an activity of a B lymphocyte antigen may differ in aminoacid sequence from the B lymphocyte antigen, such as the human B7-2sequence depicted in FIG. 8 (SEQ ID NO:2), or murine B7-2 sequencedepicted in FIG. 14 (SEQ ID NO:22), but such differences result in apeptide which functions in the same or similar manner as the Blymphocyte antigen or which has the same or similar characteristics ofthe B lymphocyte antigen. For example, a peptide having an activity ofthe B7-2 protein is defined herein as a peptide having the ability tobind to the natural ligand(s) of the B7-2 protein on immune cells, suchas CLTA4 and/or CD28 on T cells and either stimulate or inhibit immunecell costimulation. Thus, a peptide having B7-2 activity binds CTLA4and/or CD28 and stimulates or inhibits a T cell mediated immune response(as evidenced by, for example, cytokine production and/or proliferationby T cells that have received a primary activation signal). Oneembodiment provides a peptide having B7-2 binding activity, but lackingthe ability to deliver a costimulatory signal to T cells. Such a peptidecan be used to inhibit or block T cell proliferation and/or cytokinesecretion in a subject. Alternatively, a peptide having both B7-2binding activity and the ability to deliver a costimulatory signal to Tcells is used to stimulate or enhance T cell proliferation and/orcytokine secretion in a subject. Various modifications of the B7-2protein to produce these and other functionally equivalent peptides aredescribed in detail herein. The term “peptide” as used herein, refers topeptides, proteins and polypeptides.

[0075] A peptide can be produced by modification of the amino acidsequence of the human B7-2 protein shown in FIG. 8 (SEQ ID NO:2) or themurine B7-2 protein shown in FIG. 14 (SEQ ID NO:23), such as asubstitution, addition or deletion of an amino acid residue which is notdirectly involved in the function of B7-2 (i.e., the ability of B7-2 tobind CTLA4 and/or CD28 and/or stimulate or inhibit T cellcostimulation). Peptides of the invention are typically at least 20amino acid residues in length, preferably at least 40 amino acidresidues in length, and most preferably 60 amino acid residues inlength. Peptides having B7-2 activity and including at least 80 aminoacid residues in length, at least 100 amino acid residues in length, orat least 200 or more amino acid residues in length are also within thescope of the invention. A preferred peptide includes an extracellulardomain portion of the human B7-2 antigen (e.g., about amino acidresidues 24-245 of the sequence shown in FIG. 8 (SEQ ID NO:2). Otherpreferred peptides have an amino acid sequence represented by a formula:

X_(n)—Y—Z_(m)

[0076] where Y is amino acid residues selected from the group consistingof: amino acid residues 55-68 of the sequence shown in FIG. 8 (SEQ IDNO:2); amino acid residues 81-89 of the sequence shown in FIG. 8 (SEQ IDNO:2); amino acid residues 128-142 of the sequence shown in FIG. 8 (SEQID NO:2); amino acid residues 160-169 of the sequence shown in FIG. 8(SEQ ID NO:2); amino acid residues 188-200 of the sequence shown in FIG.8 (SEQ ID NO:2); and amino acid residues 269-282 of the sequence shownin FIG. 8 (SEQ ID NO:2). In the formula, X_(n) and Z_(m) are additionalamino acid residues linked to Y by an amide bond. X_(n) and Z_(m) areamino acid residues selected from amino acids contiguous to Y in theamino acid sequence shown in FIG. 8 (SEQ ID NO:2). X_(n) is amino acidresidues selected from amino acids contiguous to the amino terminus of Yin the sequence shown in FIG. 8 (SEQ ID NO:2). Z_(m) is amino acidresidues selected from amino acids contiguous to the carboxy terminus ofY in the sequence shown in FIG. 8 (SEQ ID NO:2). According to theformula, n is a number from 0 to 30 (n=0-30) and m is a number from 0 to30 (m=0-30). A particularly preferred peptide has an amino acid sequencerepresented by the formula X_(n)—Y—Z_(m), where n=0 and m=0.

[0077] Another embodiment of the invention provides a substantially purepreparation of a peptide having an activity of a novel B lymphocyteantigen such as B7-2 or B7-3. Such a preparation is substantially freeof proteins and peptides with which the peptide naturally occurs in acell or with which it naturally occurs when secreted by a cell.

[0078] The term “isolated” as used throughout this application refers toa nucleic acid, protein or peptide having an activity of a novel Blymphocyte antigen, such as B7-2, substantially free of cellularmaterial or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.An isolated nucleic acid is also free of sequences which naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the organism from which the nucleic acid is derived.

[0079] The various peptides, polypeptides, proteins, and fusion proteinsof the invention can be prepared as soluble forms. Alternatively, theproteins of the invention can be expressed on the surface of a cell,such as a CHO cell and can be prepared according to methods well knownin the art. The proteins of the invention can also be coupled to a solidphase support, such as a bead or a plate. In a specific embodiment, aB7-2Ig fusion protein is attached to a solid phase support, such as abead, for example a biodegradable bead. In a preferred embodiment, avariable region form of B7-2 is attached to a solid support. In a mostpreferred embodiment, a B7-2VIg fusion protein comprising an amino acidsequence of about position 24 to 133 of human B7-2Ig (SEQ ID NO: 2)linked to the constant domain of an IgG molecule is attached to a solidphase support.

[0080] These molecules can then be attached to a solid phase surface viaseveral possible methods. For example, the proteins of the invention,such as a variable region form of B7-2, can be crosslinked to the beadsvia covalent modification using tosyl linkage. In this method, theproteins of the invention are typically in 0.05M borate buffer, pH 9.5and added to tosyl-activated magnetic immunobeads (Dynal Inc., GreatNeck, N.Y.) according to manufacturer's instructions. After a 24 hrincubation at 22° C., the beads are collected and washed extensively. Itis not mandatory that immunomagnetic beads be used, as other methods arealso satisfactory. For example, proteins of the invention may also beimmobilized on polystyrene beads or culture vessel surfaces.

[0081] It is also possible to attach the proteins, such as B7-2VIg to asolid phase surface through an avidin- or streptavidin-biotin complex.In this particular embodiment, the soluble protein is first crosslinkedto biotin and then reacted with the solid phase surface to which avidinor streptavidin molecules are bound. It is also possible to crosslinkthe protein with avidin or streptavidin and to react these with a solidphase surface that is covered with biotin molecules.

[0082] These and other aspects of this invention are described in detailin the following subsections.

[0083] I. Isolation of Nucleic Acid from Cell Lines

[0084] Suitable cells for use in isolating nucleic acids encodingpeptides having an activity of a novel B lymphocyte antigen includecells capable of producing mRNA coding for B lymphocyte antigens (e.g.,B7-1, B7-2, B7-3) and appropriately translating the mRNA into thecorresponding protein. One source of mRNA is normal human splenic Bcells, either resting or activated by treatment with ananti-immunoglobulin antibody or an anti-MHC class II antibody, or fromsubsets of neoplastic B cells. Expression of the human B7-2 antigen isdetectable in resting B cells and in activated B cells, with mRNA levelsincreasing 4-fold from resting levels following stimulation. Totalcellular RNA can be obtained using standard techniques from resting oractivated B cells during these intervals and utilized in theconstruction of a cDNA library.

[0085] In addition, various subsets of neoplastic B cells may expressB7-2 and B7-3 and can alternatively serve as a source of the mRNA forconstruction of a cDNA library. For example, tumor cells isolated frompatients with non-Hodgkins lymphoma express B7-1 mRNA. B cells fromnodular, poorly differentiated lymphoma (NPDL), diffuse large celllymphoma (LCL) and Burkitt's lymphoma cell lines are also suitablesources of human B7-1 mRNA and, potentially B7-2 and B7-3 mRNA. Myelomasgenerally express B7-2, but not B7-1 mRNA, and, thus can provide asource of B7-2 mRNA. The Burkitf's lymphoma cell line Raji is one sourceof B lymphocyte antigen mRNA. Preferably, B7-2 mRNA is obtained from apopulation of both resting and activated normal human B cells. ActivatedB cells can be obtained by stimulation over a broad spectrum of time(e.g., from minutes to days) with, for example, an anti-immunoglobulinantibody or an anti-MCH class II antibody.

[0086] II. Isolation of mRNA and Construction of cDNA Library

[0087] Total cellular mRNA can be isolated by a variety of techniques,e.g., by using the guanidinium-thiocyanate extraction procedure ofChirgwin et al., Biochemistry 18, 5294-5299 (1979). According to thismethod, Poly(A+) mRNA is prepared and purified for use in a cDNA libraryconstruction using oligo (dT) cellulose selection. cDNA is thensynthesized from the poly(A+) RNA using oligo(dT) priming and reversetranscriptase. Moloney MLV reverse transcriptase (available fromGibco/BRL, Bethesda, Md.) or AMV reverse transcriptase (available fromSeikagaku America, Inc., St. Petersburg, Fla.) are preferably employed.

[0088] Following reverse transcription, the mRNA/DNA hybrid molecule isconverted to double stranded DNA using conventional techniques andincorporated into a suitable vector. The experiments herein employed E.coli DNA polymerase I and ribonuclease H in the conversion to doublestranded cDNA.

[0089] Cloning of the cDNAs can be accomplished using any of theconventional techniques for joining double stranded DNA with anappropriate vector. The use of synthetic adaptors is particularlypreferred, since it alleviates the possibility of cleavage of the cDNAwith restriction enzyme prior to cloning. Using this method, non-selfcomplementary, kinased adaptors are added to the DNA prior to ligationwith the vector. Virtually any adaptor can be employed. As set forth inmore detail in the examples below, non-self complementary BstXI adaptorsare preferably added to the cDNA for cloning, for ligation into a pCDM8vector prepared for cloning by digestion with BstXI.

[0090] Eucaryotic cDNA can be expressed when placed in the senseorientation in a vector that supplies an appropriate eucaryotic promoterand origin of replication and other elements including enhancers, spliceacceptors and/or donor sequences and polyadenylation signals. The cDNAsof the present invention are placed in suitable vectors containing aeucaryotic promoter, an origin of replication functional in E. coli, anSV40 origin of replication which allows growth in COS cells, and a cDNAinsertion site. Suitable vectors include πH3 (Seed and Aruffo, Proc.Natl. Acad Sci., 84:3365-3369 (1987)), πH3m (Aruffo and Seed, Proc.Natl. Acad. Sci., 84:8573-8577 (1987)), pCDM7 and pCDM8 (Seed, Nature,329:840-841 (1987), with the pCDM8 vector being particularly preferred(available commercially from Invitrogen, San Diego, Calif.).

[0091] III. Transfection of Host Cells and Screening for Novel BLymphocyte Activation Antigens

[0092] The thus prepared cDNA library is then used to clone the gene ofinterest by expression cloning techniques. A basic expression cloningtechnique has been described by Seed and Aruffo, Proc. Natl. Acad. SciUSA, 84:3365-3369 (1987) and Aruffo and Seed, Proc. Natl. Acad. Sci.USA, 84:8573-8577 (1987), although modifications to this technique maybe necessary.

[0093] According to one embodiment, plasmid DNA is introduced into asimian COS cell line (Gluzman, Cell 23:175 (1981)) by known methods oftransfection (e.g., DEAE-Dextran) and allowed to replicate and expressthe cDNA inserts. The transfectants expressing B7-1 antigen are depletedwith an anti-B7-1 monoclonal antibody (e.g., 133 and B1.1) andanti-murine IgG and IgM coated immunomagnetic beads. Transfectantsexpressing human B7-2 antigen can be positively selected by reacting thetransfectants with the fusion proteins CTLA4Ig and CD28Ig, followed bypanning with anti-human Ig antibody coated plates. Although humanCTLA4Ig and CD28Ig fusion proteins were used in the examples describedherein, given the cross-species reactivity between B7-1 and, for examplemurine B7-1, it can be expected that other fusion proteins reactive withanother cross-reactive species could be used. After panning, episomalDNA is recovered from the panned cells and transformed into a competentbacterial host, preferably E. coli. Plasmid DNA is subsequentlyreintroduced into COS cells and the cycle of expression and panningrepeated at least two times. After the final cycle, plasmid DNA isprepared from individual colonies, transfected into COS cells andanalyzed for expression of novel B lymphocyte antigens by indirectimmunofluorescence with, for example, CTLA4Ig and CD28Ig.

[0094] IV. Sequencing of Novel B Lymphocyte Antigens

[0095] Plasmids are prepared from those clones which are stronglyreactive with the CTLA4Ig and/or CD28Ig. These plasmids are thensequenced. Any of the conventional sequencing techniques suitable forsequencing tracts of DNA about 1.0 kb or larger can be employed.

[0096] As described in Example 4, a human B7-2 clone (clone29) wasobtained containing an insert of 1,120 base pairs with a single longopen reading frame of 987 nucleotides and approximately 27 nucleotidesof 3′ noncoding sequences (FIG. 8, SEQ ID NO:1). The predicted aminoacid sequence encoded by the open reading frame of the protein is shownbelow the nucleotide sequence in FIG. 8. The encoded human B7-2 protein,is predicted to be 329 amino acid residues in length (SEQ ID NO:2). Thisprotein sequence exhibits many features common to other type I Igsuperfamily membrane proteins. Protein translation is predicted to beginat the methionine codon (ATG, nucleotides 107 to 109) based on the DNAhomology in this region with the consensus eucaryotic translationinitiation site (see Kozak, M. (1987) Nucl. Acids Res. 15:8125-8148).The amino terminus of the B7-2 protein (amino acids 1 to 23) has thecharacteristics of a secretory signal peptide with a predicted cleavagebetween the alanines at positions 23 and 24 (von Heijne (1987) Nucl.Acids Res. 14:4683). Processing at this site would result in a B7-2membrane bound protein of 306 amino acids having an unmodified molecularweight of approximately 34 kDa. This protein would consist of anapproximate extracellular Ig superfamily V and C like domains of fromabout amino acid residue 24 to 245, a hydrophobic transmembrane domainof from about amino acid residue 246 to 268, and a long cytoplasmicdomain of from about amino acid residue 269 to 329. The homologies tothe Ig superfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 218. The extracellular domain also contains eight potentialN-linked glycosylation sites and, like B7-1, is probably glycosylated.Glycosylation of the human B7-2 protein may increase the molecularweight to about 50-70 kDa. The cytoplasmic domain of human B7-2, whilesomewhat longer than B7-1, contains a common region of multiplecysteines followed by positively charged amino acids which presumablyfunction as signaling or regulatory domains within an antigen-presentingcell (APC). Comparison of both the nucleotide and amino acid sequencesof the human B7-2 with the GenBank and EMBL databases yieldedsignificant homology (about 26% amino acid sequence identity) with humanB7-1. Since human B7-1, human B7-2 and murine B7-1 all bind to humanCTLA4 and CD28, the homologous amino acids probably represent thosenecessary to comprise a CTLA4 or CD28 binding sequence. E. colitransfected with a vector containing a cDNA insert encoding human B7-2(clone 29) was deposited with the American Type Culture Collection(ATCC) on Jul. 26, 1993 as Accession No. 69357.

[0097] V. Cloning Novel B Lymphocyte Antigens from Other MammalianSpecies

[0098] The present invention is not limited to human nucleic acidmolecules and contemplates that novel B lymphocyte antigen homologuesfrom other mammalian species that express B lymphocyte antigens can becloned and sequenced using the techniques described herein. B lymphocyteantigens isolated for one species (e.g., humans) which exhibitcross-species reactivity may be used to modify T cell mediated immuneresponses in a different species (e.g., mice). Isolation of cDNA clonesfrom other species can also be accomplished using human cDNA inserts,such as human B7-2 cDNA, as hybridization probes.

[0099] As described in Example 6, a murine B7-2 clone (mB7-2, clone 4)was obtained containing an insert of 1,163 base pairs with a single longopen reading frame of 927 nucleotides and approximately 126 nucleotidesof 3′ noncoding sequences (FIG. 14, SEQ ID NO:22). The predicted aminoacid sequence encoded by the open reading frame of the protein is shownbelow the nucleotide sequence in FIG. 14. The encoded murine B7-2protein, is predicted to be 309 amino acid residues in length (SEQ IDNO:23). This protein sequence exhibits many features common to othertype I Ig superfamily membrane proteins. Protein translation ispredicted to begin at the methionine codon (ATG, nucleotides 111 to 113)based on the DNA homology in this region with the consensus eucaryotictranslation initiation site (see Kozak, M. (1987) Nucl. Acids Res.15:8125-8148). The amino terminus of the murine B7-2 protein (aminoacids 1 to 23) has the characteristics of a secretory signal peptidewith a predicted cleavage between the alanine at position 23 and thevaline at position 24 (von Heijne (1987) Nucl. Acids Res. 14:4683).Processing at this site would result in a murine B7-2 membrane boundprotein of 286 amino acids having an unmodified molecular weight ofapproximately 32 kDa. This protein would consist of an approximateextracellular Ig superfamily V and C like domains of from about aminoacid residue 24 to 246, a hydrophobic transmembrane domain of from aboutamino acid residue 247 to 265, and a long cytoplasmic domain of fromabout amino acid residue 266 to 309. The homologies to the Igsuperfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 216. The extracellular domain also contains nine potentialN-linked glycosylation sites and, like murine B7-1, is probablyglycosylated. Glycosylation of the murine B7-2 protein may increase themolecular weight to about 50-70 kDa. The cytoplasmic domain of murineB7-2 contains a common region which has a cysteine followed bypositively charged amino acids which presumably functions as signalingor regulatory domain within an APC. Comparison of both the nucleotideand amino acid sequences of murine B7-2 with the GenBank and EMBLdatabases yielded significant homology (about 26% amino acid sequenceidentity) with human and murine B7-1. Murine B7-2 exhibits about 50%identity and 67% similarity with its human homologue, hB7-2. E. coli(DH106/p3) transfected with a vector (plasmid pmBx4) containing a cDNAinsert encoding murine B7-2 (clone 4) was deposited with the AmericanType Culture Collection (ATCC) on Aug. 18, 1993 as Accession No. 69388.

[0100] Nucleic acids which encode novel B lymphocyte antigens from otherspecies, such as the murine B7-2, can be used to generate eithertransgenic animals or “knock out” animals which, in turn, are useful inthe development and screening of therapeutically useful reagents. Atransgenic animal (e.g., a mouse) is an animal having cells that containa transgene, which transgene was introduced into the animal or anancestor of the animal at a prenatal, e.g., an embryonic stage. Atransgene is a DNA which is integrated into the genome of a cell fromwhich a transgenic animal develops. In one embodiment, murine B7-2 cDNAor an appropriate sequence thereof can be used to clone genomic B7-2 inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells which express B7-2protein. Methods for generating transgenic animals, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for B7-2 transgene incorporation withtissue specific enhancers, which could result in T cell costimulationand enhanced T cell proliferation and autoimmunity. Transgenic animalsthat include a copy of a B7-2 transgene introduced into the germ line ofthe animal at an embryonic stage can be used to examine the effect ofincreased B7 expression. Such animals can be used as tester animals forreagents thought to confer protection from, for example, autoimmunedisease. In accordance with this facet of the invention, an animal istreated with the reagent and a reduced incidence of the disease,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the disease.

[0101] Alternatively, the non-human homologues of B7-2 can be used toconstruct a B7-2 “knock out” animal which has a defective or alteredB7-2 gene as a result of homologous recombination between the endogenousB7-2 gene and altered B7-2 genomic DNA introduced into an embryonic cellof the animal. For example, murine B7-2 cDNA can be used to clonegenomic B7-2 in accordance with established techniques. A portion of thegenomic B7-2 DNA (e.g., such as an exon which encodes an extracellulardomain) can be deleted or replaced with another gene, such as a geneencoding a selectable marker which can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector (see e.g., Thomas, K. R. andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells arethen injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm to create a “knock out” animal. Progeny harbouring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for their ability to accept grafts, reject tumors anddefend against infectious diseases and can be used in the study of basicimmunobiology.

[0102] VI. Expression of B Lymphocyte Antigens

[0103] Host cells transfected to express peptides having the activity ofa novel B lymphocyte antigen are also within the scope of the invention.The host cell may be any procaryotic or eucaryotic cell. For example, apeptide having B7-2 activity may be expressed in bacterial cells such asE. coli, insect cells (baculovirus), yeast, or mammalian cells such asChinese hamster ovary cells (CHO) and NSO cells. Other suitable hostcells may be found in Goeddel, (1990) supra or are known to thoseskilled in the art.

[0104] For example, expression in eucaryotic cells such as mammalian,yeast, or insect cells can lead to partial or complete glycosylationand/or formation of relevant inter- or intra-chain disulfide bonds ofrecombinant protein. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari. et al, (1987) Embo J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Baculovirus vectors available for expression of proteinsin cultured insect cells (SF 9 cells) include the pAc series (Smith etal, (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M. D., (1989) Virology 170:31-39). Generally, COS cells(Gluzman, Y., (1981) Cell 23:175-182) are used in conjunction with suchvectors as pCDM8 (Seed, B., (1987) Nature 329:840) for transientamplification/expression in mammalian cells, while CHO (dhfr⁻ ChineseHamster Ovary) cells are used with vectors such as pMT2PC (Kaufman etal. (1987), EMBO J. 6:187-195) for stable amplification/expression inmammalian cells. A preferred cell line for production of recombinantprotein is the NSO myeloma cell line available from the ECACC (catalog#85110503) and described in Galfre, G. and Milstein, C. ((1981) Methodsin Enzymology 73(13):3-46; and Preparation of Monoclonal Antibodies:Strategies and Procedures, Academic Press, N.Y., N.Y.). Vector DNA canbe introduced into mammalian cells via conventional techniques such ascalcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofectin, or electroporation.Suitable methods for transforming host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989)), and other laboratory textbooks. Whenused in mammalian cells, the expression vector's control functions areoften provided by viral material. For example, commonly used promotersare derived from polyoma, Adenovirus 2, cytomegalovirus and mostfrequently, Simian Virus 40.

[0105] It is known that a small faction of cells (about 1 out of 10⁵)typically integrate DNA into their genomes. In order to identify theseintegrants, a gene that contains a selectable marker (i.e., resistanceto antibiotics) is generally introduced into the host cells along withthe gene of interest. Preferred selectable markers include those whichconfer resistance to drugs, such as G418, hygromycin and methotrexate.Selectable markers may be introduced on the same plasmid as the gene ofinterest or may be introduced on a separate plasmid. Cells containingthe gene of interest can be identified by drug selection; cells thathave incorporated the selectable marker gene will survive, while theother cells die. The surviving cells can then be screened for productionof novel B lymphocyte antigens by cell surface staining with ligands tothe B cell antigens (e.g., CTLA4Ig and CD28Ig). Alternatively, theprotein can be metabolically radiolabeled with a labeled amino acid andimmunoprecipitated from cell supernatant with an anti-B lymphocyteantigen monoclonal antibody or a fusion protein such as CTLA4Ig orCD28Ig.

[0106] Expression in procaryotes is most often carried out in E. coliwith vectors containing constitutive or inducible promoters directingthe expression of either fusion or non-fusion proteins. Fusion vectorsadd a number of amino acids usually to the amino terminus of theexpressed target gene. Such fusion vectors typically serve threepurposes: 1) to increase expression of recombinant protein; 2) toincrease the solubility of the target recombinant protein; and 3) to aidin the purification of the target recombinant protein by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the target recombinant protein to enable separation of thetarget recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne,Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-tranferase,maltose E binding protein, or protein A, respectively, to the targetrecombinant protein.

[0107]E. coli expression systems include the inducible expressionvectors pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11 (Studieret al., Gene Expression Technology. Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89; commercially available fromNovagen). In the pTrc vector system, the inserted gene is expressed witha pelB signal sequence by host RNA polymerase transcription from ahybrid trp-lac fusion promoter. After induction, the recombinant proteincan be purified from the periplasmic fraction. In the pET 11 vectorsystem, the target gene is expressed as non-fusion protein bytranscription from the T7 gn10-lac 0 fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host E. coli strains BL21 (DE3) or HMS174(DE3) from aresident λ prophage harboring a T7 gn1 under the transcriptional controlof the lacUV 5 promoter. In this system, the recombinant protein can bepurified from inclusion bodies in a denatured form and, if desired,renatured by step gradient dialysis to remove denaturants.

[0108] One strategy to maximize recombinant B7-2 expression in E. coliis to express the protein in a host bacteria with an impaired capacityto proteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy would be to alter thenucleic acid sequence of the B7-2 gene or other DNA to be inserted intoan expression vector so that the individual codons for each amino acidwould be those preferentially utilized in highly expressed E. coliproteins (Wada et al, (1992) Nuc. Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention could be carriedout by standard DNA synthesis techniques.

[0109] Novel B lymphocyte antigens and portions thereof, expressed inmammalian cells or otherwise, can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fractionation column chromatography (e.g. ion exchange, gel filtration,electrophoresis, affinity chromatography, etc.) and ultimately,crystallization (see generally, “Enzyme Purification and RelatedTechniques”, Methods in Enzymology, 22:233-577 (1971)). Once purified,partially or to homogeneity, the recombinantly produced B lymphocyteantigens or portions thereof can be utilized in compositions suitablefor pharmaceutical administration as described in detail herein.

[0110] VII. Modifications of Nucleic Acid and Amino Acid Sequences ofthe Invention and Assays for B7 Lymphocyte Antigen Activity

[0111] It will be appreciated by those skilled in the art that othernucleic acids encoding peptides having the activity of a novel Blymphocyte antigen can be isolated by the above process. Different celllines can be expected to yield DNA molecules having different sequencesof bases. Additionally, variations may exist due to geneticpolymorphisms or cell-mediated modifications of the genetic material.Furthermore, the DNA sequence of a B lymphocyte antigen can be modifiedby genetic techniques to produce proteins or peptides with altered aminoacid sequences. Such sequences are considered within the scope of thepresent invention, where the expressed peptide is capable of eitherinducing or inhibiting activated T cell mediated immune responses andimmune function.

[0112] A number of processes can be used to generate equivalents orfragments of an isolated DNA sequence. Small subregions or fragments ofthe nucleic acid encoding the B7-2 protein, for example 1-30 bases inlength, can be prepared by standard, synthetic organic chemical means.The technique is also useful for preparation of antisenseoligonucleotides and primers for use in the generation of largersynthetic fragments of B7-2 DNA.

[0113] Larger subregions or fragments of the genes encoding B lymphocyteantigens can be expressed as peptides by synthesizing the relevant pieceof DNA using the polymerase chain reaction (PCR) (Sambrook, Fritsch andManiatis, 2 Molecular Cloning, A Laboratory Manual, Cold Spring Harbor,N.Y., (1989)), and ligating the thus obtained DNA into an appropriateexpression vector. Using PCR, specific sequences of the cloned doublestranded DNA are generated, cloned into an expression vector, and thenassayed for CTLA4/CD28 binding activity. For example, to express asecreted (soluble) form of the human B7-2 protein, using PCR, a DNA canbe synthesized which does not encode the transmembrane and cytoplasmicregions of the protein. This DNA molecule can be ligated into anappropriate expression vector and introduced into a host cell such asCHO, where the B7-2 protein fragment is synthesized and secreted. TheB7-2 protein fragment can then readily be obtained from the culturemedia.

[0114] In another embodiment, mutations can be introduced into a DNA byany one of a number of methods, including those for producing simpledeletions or insertions, systematic deletions, insertions orsubstitutions of clusters of bases or substitutions of single bases, togenerate variants or modified equivalents of B lymphocyte antigen DNA.For example, changes in the human B7-2 cDNA sequence shown in FIG. 8(SEQ ID NO:1) or murine B7-2 cDNA sequence shown in FIG. 14 (SEQ IDNO:22) such as amino acid substitutions or deletions are preferablyobtained by site-directed mutagenesis. Site directed mutagenesis systemsare well known in the art. Protocols and reagents can be obtainedcommercially from Amersham International PLC, Amersham, U.K.

[0115] Peptides having an activity of a novel B lymphocyte antigen,i.e., the ability to bind to the natural ligand(s) of a B lymphocyteantigen on T cells and either stimulate (amplify) or inhibit (block)activated T cell mediated immune responses, as evidenced by, forexample, cytokine production and/or T cell proliferation by T cells thathave received a primary activation signal are considered within thescope of the invention. More specifically, peptides that bind to Tlymphocytes, for example CD28⁺ cells, may be capable of delivering acostimulatory signal to the T lymphocytes, which, when transmitted inthe presence of antigen and class II MHC, or other material capable oftransmitting a primary signal to the T cell, results in activation ofcytokine genes within the T cell. Alternatively, such a peptide can beused in conjunction with class I MHC to thereby activate CD8⁺ cytolyticT cells. In addition, soluble, monomeric forms of the B7-2 protein, mayretain the ability to bind to their natural ligand(s) on CD28⁺ T cellsbut, perhaps because of insufficient cross-linking with the ligand, failto deliver the secondary signal essential for enhanced cytokineproduction and cell division. Such peptides, which provide a means toinduce a state of anergy or tolerance in the cells, are also consideredwithin the scope of the invention.

[0116] Screening the peptides for those which retain a characteristic Blymphocyte antigen activity as described herein can be accomplishedusing one or more of several different assays. For example, the peptidescan be screened for specific reactivity with an anti-B7-2 monoclonalantibody reactive with cell surface B7-2 or with a fusion protein, suchas CTLA4Ig or CD28Ig. Specifically, appropriate cells, such as COScells, can be transfected with a B7-2 DNA encoding a peptide and thenanalyzed for cell surface phenotype by indirect immunofluorescence andflow cytometry to determine whether the peptide has B7-2 activity. Cellsurface expression of the transfected cells is evaluated using amonoclonal antibody specifically reactive with cell surface B7-2 or witha CTLA4Ig or CD28Ig fusion protein. Production of secreted forms of B7-2is evaluated using anti-B7-2 monoclonal antibody or CTLA4Ig or CD28fusion protein for immunoprecipitation.

[0117] Other, more preferred, assays take advantage of the functionalcharacteristics of the B7-2 antigen. As previously set forth, theability of T cells to synthesize cytokines depends not only on occupancyor cross-linking of the T cell receptor for antigen (the “primaryactivation signal” provided by, for example anti-CD3, or phorbol esterto produce an “activated T cell”), but also on the induction of acostimulatory signal, in this case, by interaction with a B lymphocyteantigen, such as B7-2, B7-1 or B7-3. The binding of B7-2 to its naturalligand(s) on, for example, CD28⁺ T cells, has the effect of transmittinga signal to the T cell that induces the production of increased levelsof cytokines, particularly of interleukin-2, which in turn stimulatesthe proliferation of the T lymphocytes. Other assays for B7-2 functionthus involve assaying for the synthesis of cytokines, such asinterleukin-2, interleukin-4 or other known or unknown novel cytokines,and/or assaying for T cell proliferation by CD28⁺ T cells which havereceived a primary activation signal.

[0118] In vitro, T cells can be provided with a first or primaryactivation signal by anti-T3 monoclonal antibody (e.g. anti-CD3) orphorbol ester or, more preferably, by antigen in association with classII MHC. T cells which have received a primary activation signal arereferred to herein as activated T cells. B7-2 function is assayed byadding a source of B7-2 (e.g., cells expressing a peptide having B7-2activity or a secreted form of B7-2) and a primary activation signalsuch as antigen in association with Class II MHC to a T cell culture andassaying the culture supernatant for interleukin-2, gamma interferon, orother known or unknown cytokine. For example, any one of severalconventional assays for interleukin-2 can be employed, such as the assaydescribed in Proc. Natl. Acad. Sci. USA, 86:1333 (1989) the pertinentportions of which are incorporated herein by reference. A kit for anassay for the production of interferon is also available from GenzymeCorporation (Cambridge, Mass.). T cell proliferation can also bemeasured as described in the Examples below. Peptides that retain thecharacteristics of the B7-2 antigen as described herein may result inincreased per cell production of cytokines, such as IL-2, by T cells andmay also result in enhanced T cell proliferation when compared to anegative control in which a costimulatory signal is lacking.

[0119] The same basic functional assays can also be used to screen forpeptides having B7-2 activity, but which lack the ability to deliver acostimulatory signal, but in the case of such peptides, addition of theB7-2 protein will not result in a marked increase in proliferation orcytokine secretion by the T cells. The ability of such proteins toinhibit or completely block the normal B7-2 costimulatory signal andinduce a state of anergy can be determined using subsequent attempts atstimulation of the T cells with antigen presenting cells that expresscell surface B7-2 and present antigen. If the T cells are unresponsiveto the subsequent activation attempts, as determined by IL-2 synthesisand T cell proliferation, a state of anergy has been induced. See, e.g.,Gimmi, C. D. et al. (1993) Proc. Natl. Acad. Sci. USA 9, 6586-6590; andSchwartz (1990) Science, 248, 1349-1356, for assay systems that can usedas the basis for an assay in accordance with the present invention.

[0120] It is possible to modify the structure of a peptide having theactivity of a novel B lymphocyte antigen for such purposes as increasingsolubility, enhancing therapeutic or prophylactic efficacy, or stability(e.g., shelf life ex vivo and resistance to proteolytic degradation invivo). Such modified peptides are considered function al equivalents ofthe B lymphocyte antigens as defined herein. For example, a peptidehaving B7-2 activity can be modified so that it maintains the ability toco-stimulate T cell proliferation and/or produce cytokines. Thoseresidues shown to be essential to interact with the CTLA4/CD28 receptorson T cells can be modified by replacing the essential amino acid withanother, preferably similar amino acid residue (a conservativesubstitution) whose presence is shown to enhance, diminish, but noteliminate, or not effect receptor interaction. In addition, those aminoacid residues which are not essential for receptor interaction can bemodified by being replaced by another amino acid whose incorporation mayenhance, diminish, or not effect reactivity.

[0121] Another example of modification of a peptide having the activityof a novel B lymphocyte antigen is substitution of cysteine residuespreferably with alanine, serine, threonine, leucine or glutamic acidresidues to minimize dimerization via disulfide linkages. In addition,amino acid side chains of a peptide having B7-2 activity can bechemically modified. Another modification is cyclization of the peptide.

[0122] In order to enhance stability and/or reactivity, peptides havingB7-2 activity can be modified to incorporate one or more polymorphismsin the amino acid sequence of the antigen resulting from any naturalallelic variation. Additionally, D-amino acids, non-natural amino acids,or non-amino acid analogs can be substituted or added to produce amodified protein within the scope of this invention. Furthermore, thepeptides can be modified using polyethylene glycol (PEG) according tothe method of A. Sehon and co-workers (Wie et al., supra) to produce apeptide conjugated with PEG. In addition, PEG can be added duringchemical synthesis of the peptide. Other modifications of the peptidesinclude reduction/alkylation (Tarr in: Methods of ProteinMicrocharacterization, J. E. Silver ed., Humana Press, Clifton N.J.155-194 (1986)); acylation (Tarr, supra); chemical coupling to anappropriate carrier (Mishell and Shiigi, eds, Selected Methods inCellular Immunology, W H Freeman, San Francisco, Calif. (1980), U.S.Pat. No. 4,939,239; or mild formalin treatment (Marsh (1971), Int. Arch.of Allergy and Appl. Immunol. 41:199-215).

[0123] To facilitate purification and potentially increase solubility ofa peptide, it is possible to add an amino acid fusion moiety to theprotein backbone. For example, hexa-histidine can be added to thepeptide for purification by immobilized metal ion affinitychromatography (Hochuli, E. et al., (1988) Bio/Technology 6:1321-1325).In addition, to facilitate isolation of a B lymphocyte antigen free ofirrelevant sequences, specific endoprotease cleavage sites can beintroduced between the sequences of a fusion moiety and the peptide. Itmay be necessary to increase the solubility of a peptide by addingfunctional groups to the peptide, or by omitting hydrophobic regions ofthe peptide.

[0124] VII. Uses of Nucleic Acid Sequences Encoding B LymphocyteAntigens and Peptides having B7-2 Activity

[0125] A. Molecular Probes

[0126] The nucleic acids of this invention are useful diagnostically,for tracking the progress of disease, by measuring the activation statusof B lymphocytes in biological samples or for assaying the effect of amolecule on the expresssion of a B lymphocyte antigen (e.g., detectingcellular mRNA levels). In accordance with these diagnostic assays, thenucleic acid sequences are labeled with a detectable marker, e.g., aradioactive, fluorescent, or biotinylated marker and used in aconventional dot blot or Northern hybridization procedure to probe mRNAmolecules of total or poly(A+) RNAs from a biological sample.

[0127] B. Antibody Production

[0128] The peptides and fusion proteins produced from the nucleic acidmolecules of the present invention can also be used to produceantibodies specifically reactive with B lymphocyte antigens. Forexample, by using a full-length B7-2 protein, or a peptide fragmentthereof, having an amino acid sequence based on the predicted amino acidsequence of B7-2, anti-protein/anti-peptide polyclonal antisera ormonoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the protein or peptide which elicits an antibody response in themammal. The immunogen can be, for example, a recombinant B7-2 protein,or fragment thereof, a synthetic peptide fragment or a cell thatexpresses a B lymphocyte antigen on its surface. The cell can be forexample, a splenic B cell or a cell transfected with a nucleic acidencoding a B lymphocyte antigen of the invention (e.g., a B7-2 cDNA)such that the B lymphocyte antigen is expressed on the cell surface. Theimmunogen can be modified to increase its immunogenicity. For example,techniques for conferring immunogenicity on a peptide includeconjugation to carriers or other techniques well known in the art. Forexample, the peptide can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassay can beused with the immunogen as antigen to assess the levels of antibodies.

[0129] Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera. To produce monoclonalantibodies, antibody producing cells (lymphocytes) can be harvested froman immunized animal and fused with myeloma cells by standard somaticcell fusion procedures thus immortalizing these cells and yieldinghybridoma cells. Such techniques are well known in the art. For example,the hybridoma technique originally developed by Kohler and Milstein(Nature (1975) 256:495-497) as well as other techniques such as thehuman B-cell hybridoma technique (Kozbar et al., Immunol. Today (1983)4:72), the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985)(Allen R. Bliss, Inc., pages 77-96), and screening of combinatorialantibody libraries (Huse et al., Science (1989) 246:1275). Hybridomacells can be screened immunochemically for production of antibodiesspecifically reactive with the peptide and monoclonal antibodiesisolated.

[0130] The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with a peptide having theactivity of a novel B lymphocyte antigen or fusion protein as describedherein. Antibodies can be fragmented using conventional techniques andthe fragments screened for utility in the same manner as described abovefor whole antibodies. For example, F(ab′)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab′)₂ fragment can betreated to reduce disulfide bridges to produce Fab′ fragments. Theantibody of the present invention is further intended to includebispecific and chimeric molecules having an anti-B lymphocyte antigen(i.e., B7-2, B7-3) portion.

[0131] Particularly preferred antibodies are anti-human B7-2 monoclonalantibodies produced by hybridomas HA3.1F9, HA5.2B7 and HF2.3D1. Thepreparation and characterization of these antibodies is described indetail in Example 8. Monoclonal antibody HA3.1F9 was determined to be ofthe IgG1 isotype; monoclonal antibody HA5.2B7 was determined to be ofthe IgG2b isotype; and monoclonal anibody HF2.3D1 was determined to beof the IgG2a isotype. Hybidoma cells were deposited with the AmericanType Culture Collection, which meets the requirements of the BudapestTreaty, on Jul. 19, 1994 as ATCC Accession No. (hybridoma HA3.1F9), ATCCAccession No. (HA5.2B7) and ATCC Accession No. (HF2.3D1).

[0132] When antibodies produced in non-human subjects are usedtherapeutically in humans, they are recognized to varying degrees asforeign and an immune response may be generated in the patient. Oneapproach for minimizing or eliminating this problem, which is preferableto general immunosuppression, is to produce chimeric antibodyderivatives, i.e., antibody molecules that combine a non-human animalvariable region and a human constant region. Chimeric antibody moleculescan include, for example, the antigen binding domain from an antibody ofa mouse, rat, or other species, with human constant regions. A varietyof approaches for making chimeric antibodies have been described and canbe used to make chimeric antibodies containing the immunoglobulinvariable region which recognizes the gene product of the novel Blymphocyte antigens of the invention. See, for example, Morrison et al.,Proc. Natl. Acad Sci. U.S.A. 81:6851 (1985); Takeda et al., Nature314:452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al.,U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent PublicationEP171496; European Patent Publication 0173494, United Kingdom Patent GB2177096B. It is expected that such chimeric antibodies would be lessimmunogenic in a human subject than the corresponding non-chimericantibody.

[0133] For human therapeutic purposes, the monoclonal or chimericantibodies specifically reactive with a peptide having the activity of aB lymphocyte antigen as described herein can be further humanized byproducing human variable region chimeras, in which parts of the variableregions, especially the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. General reviews of “humanized” chimericantibodies are provided by Morrison, S. L. (1985) Science 229:1202-1207and by Oi et al. (1986) BioTechniques 4:214. Such altered immunoglobulinmolecules may be made by any of several techniques known in the art,(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312 (1983);Kozbor et al., Immunology Today, 4:7279 (1983); Olsson et al., Meth.Enzymol., 92:3-16 (1982)), and are preferably made according to theteachings of PCT Publication WO92/06193 or EP 0239400. Humanizedantibodies can be commercially produced by, for example, ScotgenLimited, 2 Holly Road, Twickenham, Middlesex, Great Britain. Suitable“humanized” antibodies can be alternatively produced by CDR or CEAsubstitution (see U.S. Pat. No. 5,225,539 to Winter; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060). Humanized antibodieswhich have reduced immunogenicity are preferred for immunotherapy inhuman subjects. Immunotherapy with a humanized antibody will likelyreduce the necessity for any concomitant immunosuppression and mayresult in increased long term effectiveness for the treatment of chronicdisease situations or situations requiring repeated antibody treatments.

[0134] As an alterntive to humanizing a monoclonal antibody from a mouseor other species, a human monoclonal antibody directed against a humanprotein can be generated. Transgenic mice carrying human antibodyrepertoires have been created which can be immunized with a human Blymphocyte antigen, such as B7-2. Splenocytes from these immunizedtransgenic mice can then be used to create hybridomas that secrete humanmonoclonal antibodies specifically reactive with a human B lymphocyteantigen (see, e.g., Wood et al. PCT publication WO 91/00906,Kucherlapati et al. PCT publication WO 91/10741; Lonberg et al. PCTpublication WO 92/03918; Kay et al. PCT publication 92/03917; Lonberg,N. et al. (1994) Nature 3:856-859; Green, L. L. et al. (1994) NatureGenet. 7:13-21; Morrison, S. L. et al. (1994) Proc. Natl. Acad. Sci. USA81:6851-6855; Bruggeman et al. (1993) Year Immunol 7:33-40; Tuaillon etal. (1993) PNAS 90:3720-3724; and Bruggeman et al. (1991) Eur J Immunol21:1323-1326).

[0135] Monoclonal antibody compositions of the invention can also beproduced by other methods well known to those skilled in the art ofrecombinant DNA technology. An alternative method, referred to as the“combinatorial antibody display” method, has been developed to identifyand isolate antibody fragments having a particular antigen specificity,and can be utilized to produce monoclonal antibodies that bind a Blymphocyte antigen of the invention (for descriptions of combinatorialantibody display see e.g., Sastry et al. (1989) PNAS 86:5728; Huse etal. (1989) Science 246:1275; and Orlandi et al. (1989) PNAS 86:3833).After immunizing an animal with a B lymphocyte antigen, the antibodyrepertoire of the resulting B-cell pool is cloned. Methods are generallyknown for directly obtaining the DNA sequence of the variable regions ofa diverse population of immunoglobulin molecules by using a mixture ofoligomer primers and PCR. For instance, mixed oligonucleotide primerscorresponding to the 5′ leader (signal peptide) sequences and/orframework 1 (FR1) sequences, as well as primer to a conserved 3′constant region primer can be used for PCR amplification of the heavyand light chain variable regions from a number of murine antibodies(Larrick et al. (1991) Biotechniques 11:152-156). A similar strategy canalso been used to amplify human heavy and light chain variable regionsfrom human antibodies (Larrick et al. (1991) Methods: Companion toMethods in Enzymology 2:106-110).

[0136] In an illustrative embodiment, RNA is isolated from activated Bcells of, for example, peripheral blood cells, bone marrow, or spleenpreparations, using standard protocols (e.g., U.S. Pat. No. 4,683,202;Orlandi, et al. PNAS (1989) 86:3833-3837; Sastry et al., PNAS (1989)86:5728-5732; and Huse et al. (1989) Science 246:1275-1281.)First-strand cDNA is synthesized using primers specific for the constantregion of the heavy chain(s) and each of the κ and λ light chains, aswell as primers for the signal sequence. Using variable region PCRprimers, the variable regions of both heavy and light chains areamplified, each alone or in combinantion, and ligated into appropriatevectors for further manipulation in generating the display packages.Oligonucleotide primers useful in amplification protocols may be uniqueor degenerate or incorporate inosine at degenerate positions.Restriction endonuclease recognition sequences may also be incorporatedinto the primers to allow for the cloning of the amplified fragment intoa vector in a predetermined reading frame for expression.

[0137] The V-gene library cloned from the immunization-derived antibodyrepertoire can be expressed by a population of display packages,preferably derived from filamentous phage, to form an antibody displaylibrary. Ideally, the display package comprises a system that allows thesampling of very large diverse antibody display libraries, rapid sortingafter each affinity separation round, and easy isolation of the antibodygene from purified display packages. In addition to commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZIP™ phage display kit, catalog no. 240612), examplesof methods and reagents particularly amenable for use in generating adiverse antibody display library can be found in, for example, Ladner etal. U.S. Pat. No. 5,223,409; Kang et al. International Publication No.WO 92/18619; Dower et al. International Publication No. WO 91/17271;Winter et al. International Publication WO 92/20791; Markland et al.International Publication No. WO 92/15679; Breitling et al.International Publication WO 93/01288; McCafferty et al. InternationalPublication No. WO 92/01047; Garrard et al. International PublicationNo. WO 92/09690; Ladner et al. International Publication No. WO90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al.(1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffths et al. (1993) EMBO J. 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 3:624-628;Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991)Bio/Technology 2:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.

[0138] In certain embodiments, the V region domains of heavy and lightchains can be expressed on the same polypeptide, joined by a flexiblelinker to form a single-chain Fv fragment, and the scFV genesubsequently cloned into the desired expression vector or phage genome.As generally described in McCafferty et al., Nature (1990) 348:552-554,complete V_(H) and V_(L) domains of an antibody, joined by a flexible(Gly₄-Ser)₃ linker can be used to produce a single chain antibody whichcan render the display package separable based on antigen affinity.Isolated scFV antibodies immunoreactive with a peptide having activityof a B lymphocyte antigen can subsequently be formulated into apharmaceutical preparation for use in the subject method.

[0139] Once displayed on the surface of a display package (e.g.,filamentous phage), the antibody library is screened with a B lymphocyteantigen protein, or peptide fragment thereof, to identify and isolatepackages that express an antibody having specificity for the Blymphocyte antigen. Nucleic acid encoding the selected antibody can berecovered from the display package (e.g., from the phage genome) andsubcloned into other expression vectors by standard recombinant DNAtechniques.

[0140] The antibodies of the current invention can be usedtherapeutically to inhibit T cell activation through blockingreceptor:ligand interactions necessary for costimulation of the T cell.These so-called “blocking antibodies” can be identified by their abilityto inhibit T cell proliferation and/or cytokine production when added toan in vitro costimulation assay as described herein. The ability ofblocking antibodies to inhibit T cell function s may result inimmunosuppression and/or tolerance when these antibodies areadministered in vivo.

[0141] C. Protein Purification

[0142] The polyclonal or monoclonal antibodies of the current invention,such as an antibody specifically reactive with a recombinant orsynthetic peptide having B7-2 activity or B7-3 activity can also be usedto isolate the native B lymphocyte antigen from cells. For example,antibodies reactive with the peptide can be used to isolate thenaturally-occurring or native form of B7-2 from activated B lymphocytesby immunoaffinity chromatography. In addition, the native form of B7-3can be isolated from B cells by immunoaffinity chromatography withmonoclonal antibody BB-1.

[0143] D. Other Therapeutic Reagents

[0144] The nucleic acid sequences and novel B lymphocyte antigensdescribed herein can be used in the development of therapeutic reagentshaving the ability to either upregulate (e.g., amplify) or downregulate(e.g., suppress or tolerize) T cell mediated immune responses. Forexample, peptides having B7-2 activity, including soluble, monomericforms of the B7-2 antigen or a B7-2 fusion protein, e.g., B7-2Ig, andanti-B7-2 antibodies that fail to deliver a costimulatory signal to Tcells that have received a primary activation signal, can be used toblock the B7-2 ligand(s) on T cells and thereby provide a specific meansby which to cause immunosuppression and/or induce tolerance in asubject. Such blocking or inhibitory forms of B lymphocyte antigens andfusion proteins and blocking antibodies can be identified by theirability to inhibit T cell proliferation and/or cytokine production whenadded to an in vitro costimulation assay as previously described herein.In contrast to the monomeric form, stimulatory forms of B7-2, such as anintact cell surface B7-2, retain the ability to transmit thecostimulatory signal to the T cells, resulting in an increased secretionof cytokines when compared to activated T cells that have not receivedthe secondary signal.

[0145] In addition, fusion proteins comprising a first peptide having anactivity of B7-2 fused to a second peptide having an activity of anotherB lymphocyte antigen (e.g., B7-1) can be used to modify T cell mediatedimmune responses. Alternatively, two separate peptides having anactivity of B lymphocyte antigens, for example, B7-2 and B7-1, or acombination of blocking antibodies (e.g., anti-B7-2 and anti-B7-1monoclonal antibodies) can be combined as a single composition oradministered separately (simultaneously or sequentially), to upregulateor downregulate T cell mediated immune responses in a subject.Furthermore, a therapeutically active amount of one or more peptideshaving B7-2 activity and or B7-1 activity can be used in conjunctionwith other immunomodulating reagents to influence immune responses.Examples of other immunomodulating reagents include blocking antibodies,e.g., against CD28 or CTLA4, against other T cell markers or againstcytokines, fusion proteins, e.g., CTLA4Ig, or immunosuppressive drugs,e.g., cyclosporine A or FK506.

[0146] The peptides produced from the nucleic acid molecules of thepresent invention may also be useful in the construction of therapeuticagents which block T cell function by destruction of the T cell. Forexample, as described, secreted forms of a B lymphocyte antigen can beconstructed by standard genetic engineering techniques. By linking asoluble form of B7-1, B7-2 or B7-3 to a toxin such as ricin, an agentcapable of preventing T cell activation can be made. Infusion of one ora combination of immunotoxins, e.g., B7-2-ricin, B7-1-ricin, into apatient may result in the death of T cells, particularly of activated Tcells that express higher amounts of CD28 and CTLA4. Soluble forms ofB7-2 in a monovalent form alone may be useful in blocking B7-2 function,as described above, in which case a carrier molecule may also beemployed.

[0147] Another method of preventing the function of a B lymphocyteantigen is through the use of an antisense or triplex oligonucleotide.For example, an oligonucleotide complementary to the area around theB7-1, B7-2 or B7-3 translation initiation site, (e.g., for B7-1,TGGCCCATGGCTTCAGA, (SEQ ID NO:20) nucleotides 326-309 and for B7-2,GCCAAAATGGATCCCCA (SEQ ID NO:21)), can be synthesized. One or moreantisense oligonucleotides can be added to cell media, typically at 200μg/ml, or administered to a patient to prevent the synthesis of B7-1,B7-2 and/or B7-3. The antisense oligonucleotide is taken up by cells andhybridizes to the appropriate B lymphocyte antigen mRNA to preventtranslation. Alternatively, an oligonucleotide which bindsdouble-stranded DNA to form a triplex construct to prevent DNA unwindingand transcription can be used. As a result of either, synthesis of oneor more B lymphocyte antigens is blocked.

[0148] In a specific embodiment, T cells are obtained from a subject andcultured ex vivo to expand the population of T cells. In a furtherembodiment the T cells are then administered to a subject. T cells canbe stimulated to proliferate in vitro by, for example, providing to theT cells a primary activation signal and a costimulatory signal, asdescribed in detail in the Examples section. A preferred costimulatorymolecule for stimulating proliferation of activated T cells, such as Tcells stimulated through their T cell receptor, is aB7-2VIg fusionprotein. However, other forms of B7-2Ig fusion proteins can also be usedto costimulate proliferation of T cells. In a specific embodiment of theinvention, activated T cells are costimulated with a B7-2VIg protein,such that a response by the activated T cells is stimulated. In oneembodiment T cells are cultured ex vivo according to the methoddescribed in PCT Application No. WO 94/29436, using B7-21g, or morepreferably B7-2VIg as the costimulatory molecule. The costimulatorymolecule can be soluble, attached to acell membrane or attached to asolid surface, such as a bead. In a preferred embodiment, a Thelper-type 2 (Th2) response is preferentially stimulated.

[0149] E. Therapeutic uses by Downregulation of Immune Responses

[0150] Given the structure and function of the novel B lymphocyteantigens disclosed herein, it is possible to downregulate the functionof a B lymphocyte antigen, and thereby downregulate immune responses, ina number of ways. Downregulation may be in the form of inhibiting orblocking an immune response already in progress or may involvepreventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

[0151] Downregulating or preventing one or more B lymphocyte antigenfunctions, e.g., preventing high level lymphokine synthesis by activatedT cells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this manner prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toacheive sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens. For example, it may be desirable to block the function of B7-2and B7-1, B7-2 and B7-3, B7-1 and B7-3 by administering a soluble formof a combination of peptides having an activity of each of theseantigens or a blocking antibody (separately or together in a singlecomposition) prior to transplantation. Alternatively, inhibitory formsof B lymphocyte antigens can be used with other suppressive agents suchas blocking antibodies against other T cell markers or againstcytokines, other fusion proteins, e.g., CTLA4Ig, or immunosuppressivedrugs.

[0152] The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. The function ally importantaspects of B7-1 are conserved structurally between species and it istherefore likely that other B lymphocyte antigens can function acrossspecies, thereby allowing use of reagents composed of human proteins inanimal systems. Examples of appropriate systems which can be usedinclude allogeneic cardiac grafts in rats and xenogeneic pancreaticislet cell grafts in mice, both of which have been used to examine theimmunosuppressive effects of CTLA4Ig fusion proteins in vivo asdescribed in Lenschow et al., Science, 257: 789-792 (1992) and Turka etal., Proc. Natl. Acad. Sci. USA, 89: 11102-11105 (1992). In addition,murine models of GVHD (see Paul ed., Fundamental Immunology, RavenPress, New York, 1989, pp. 846-847) can be used to determine the effectof blocking B lymphocyte antigen function in vivo on the development ofthat disease.

[0153] Blocking B lymphocyte antigen function, e.g., by use of a peptidehaving B7-2 activity alone or in combination with a peptide having B7-1activity and/or a peptide having B7-3 activity, may also betherapeutically useful for treating autoimmune diseases. Many autoimmunedisorders are the result of inappropriate activation of T cells that arereactive against self tissue and which promote the production ofcytokines and autoantibodies involved in the pathology of the diseases.Preventing the activation of autoreactive T cells may reduce oreliminate disease symptoms. Administration of reagents which blockcostimulation of T cells by disrupting receptor:ligand interactions of Blymphocyte antigens can be used to inhibit T cell activation and preventproduction of autoantibodies or T cell-derived cytokines which may beinvolved in the disease process. Additionally, blocking reagents mayinduce antigen-specific tolerance of autoreactive T cells which couldlead to long-term relief from the disease. The efficacy of blockingreagents in preventing or alleviating autoimmune disorders can bedetermined using a number of well-characterized animal models of humanautoimmune diseases. Examples include murine experimental autoimmuneencephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZBhybrid mice, murine autoimmune collagen arthritis, diabetes mellitus inNOD mice and BB rats, and murine experimental myasthenia gravis (seePaul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.840-856).

[0154] The IgE antibody response in atopic allergy is highly T celldependent and, thus, inhibition of B lymphocyte antigen induced T cellactivation may be useful therapeutically in the treatment of allergy andallergic reactions. An inhibitory form of B7-2 protein, such as apeptide having B7-2 activity alone or in combination with a peptidehaving the activity of another B lymphocyte antigen, such as B7-1, canbe administered to an allergic subject to inhibit T cell mediatedallergic responses in the subject. Inhibition of B lymphocyte antigencostimulation of T cells may be accompagnied by exposure to allergen inconjunction with appropriate MHC molecules. Allergic reactions may besystemic or local in nature, depending on the route of entry of theallergen and the pattern of deposition of IgE on mast cells orbasophils. Thus, it may be necessary to inhibit T cell mediated allergicresponses locally or systemically by proper administration of aninhibitory form of B7-2 protein.

[0155] Inhibition of T cell activation through blockage of B lymphocyteantigen function may also be important therapeutically in viralinfections of T cells. For example, in the acquired immune deficiencysyndrome (AIDS), viral replication is stimulated by T cell activation.Blocking B7-2 function could lead to a lower level of viral replicationand thereby ameliorate the course of AIDS. In addition, it may also benecessary to block the function of a combination of B lymphocyteantigens i.e., B7-1, B7-2 and B7-3. Surprisingly, HTLV-I infected Tcells express B7-1 and B7-2. This expression may be important in thegrowth of HTLV-I infected T cells and the blockage of B7-1 functiontogether with the function of B7-2 and/or B7-3 may slow the growth ofHTLV-I induced leukemias. Alternatively, stimulation of viralreplication by T cell activation may be induced by contact with astimulatory form of B7-2 protein, for such purposes as generatingretroviruses (e.g., various HIV isolates) in sufficient quantities forisolatation and use.

[0156] F. Therapeutic uses by Upregulation of Immune Responses

[0157] Upregulation of a B lymphocyte antigen function, as a means ofupregulating immune responses, may also be useful in therapy.Upregulation of immune responses may be in the form of enhancing anexisting immune response or eliciting an initial immune response. Forexample, enhancing an immune response through stimulating B lymphocyteantigen function may be useful in cases of viral infection. Viralinfections are cleared primarily by cytolytic T cells. In accordancewith the present invention, it is believed that B7-2 and thus, B7-1 andB7-3 with their natural ligand(s) on T cells may result in an increasein the cytolytic activity of at least some T cells. It is also believedthat B7-2, B7-1, and B7-3 are involved in the initial activation andgeneration of CD8+ cytotoxic T cells. The addition of a soluble peptidehaving B7-2 activity, alone, or in combination with a peptide having theactivity of another B lymphocyte antigen, in a multi-valent form, tostimulate T cell activity through the costimulation pathway would thusbe therapeutically useful in situations where more rapid or thoroughclearance of virus would be beneficial. These would include viral skindiseases such as Herpes or shingles, in which cases the multi-valentsoluble peptide having B7-2 activity or combination of such peptideand/or a peptide having B7-1 activity and/or a peptide having B7-3activity is delivered topically to the skin. In addition, systemic viraldiseases such as influenza, the common cold, and encephalitis might bealleviated by the administration of stimulatory forms of B lymphocyteantigens systemically.

[0158] Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide having B7-2 activity (alone or in combination with a peptidehaving B7-1 activity and/or a peptide having B7-3 activity) or togetherwith a stimulatory form of a soluble peptide having B7-2 activity (aloneor in combination with a peptide having B7-1 activity and/or a peptidehaving B7-3 activity) and reintroducing the in vitro activated T cellsinto the patient. Another method of enhancing anti-viral immuneresponses would be to isolate infected cells from a patient, transfectthem with a nucleic acid encoding a peptide having the activity of a Blymphocyte antigen as described herein such that the cells express allor a portion of a B lymphocyte antigen on their surface, e.g., B7-2 orB7-3, and reintroduce the transfected cells into the patient. Theinfected cells would now be capable of delivering a costimulatory signalto, and thereby activate, T cells in vivo.

[0159] Stimulatory forms of B lymphocyte antigens may also be usedprophylactically in vaccines against various pathogens. Immunity againsta pathogen, e.g., a virus, could be induced by vaccinating with a viralprotein along with a stimulatory form of a peptide having B7-2 activityor another peptide having the activity of B lymphocyte antigen in anappropriate adjuvant. Alternately, an expression vector which encodesgenes for both a pathogenic antigen and a peptide having the activity ofa B lymphocyte antigen, e.g., a vaccinia virus expression vectorengineered to express a nucleic acid encoding a viral protein and anucleic acid encoding a peptide having B7-2 activity as describedherein, can be used for vaccination. Presentation of B7-2 with class IMHC proteins by, for example, a cell transfected to coexpress a peptidehaving B7-2 activity and MHC class I α chain protein and β₂microglobulin may also result in activation of cytolytic CD8+ T cellsand provide immunity from viral infection. Pathogens for which vaccinesmay be useful include hepatitis B, hepatitis C, Epstein-Barr virus,cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria andschistosomiasis.

[0160] In another application, upregulation or enhancement of Blymphocyte antigen function may be useful in the induction of tumorimmunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,neuroblastoma, carcinoma) transfected with a nucleic acid encoding atleast one peptide having the activity of a B lymphocyte antigen, such asB7-2, can be administered to a subject to overcome tumor-specifictolerance in the subject. If desired, the tumor cell can be transfectedto express a combination of peptides having the activity of a number ofB lymphocyte antigens (e.g., B7-1, B7-2, B7-3). For example, tumor cellsobtained from a patient can be transfected ex vivo with an expressionvector directing the expression of a peptide having B7-2 activity alone,or in conjuction with a peptide having B7-1 activity and/or B7-3activity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

[0161] The presence of the peptide having the activity of a B lymphocyteantigen(s) on the surface of the tumor cell provides the necessarycostimulation signal to T cells to induce a T cell mediated immuneresponse against the transfected tumor cells. In addition, tumor cellswhich lack MHC class I or MHC class II molecules, or which fail toexpress sufficient amounts of MHC class I or MHC class II molecules, canbe transfected with nucleic acid encoding all or a portion of (e.g., acytoplasmic-domain truncated portion) of an MHC class I α chain proteinand β₂ microglobulin protein or an MHC class II a chain protein and anMHC class II β chain protein to thereby express MHC class I or MHC classII proteins on the cell surface. Expression of the appropriate class Ior class II MHC in conjunction with a peptide having the activity of a Blymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediatedimmune response against the transfected tumor cell. Optionally, a geneencoding an antisense construct which blocks expression of an MHC classII associated protein, such as the invariant chain, can also becotransfected with a DNA encoding a peptide having the activity of a Blymphocyte antigen to promote presentation of tumor associated antigensand induce tumor specific immunity. Expression of B7-1 by B7 negativemurine tumor cells has been shown to induce T cell mediated specificimmunity accompanied by tumor rejection and prolonged protection totumor challenge in mice (Chen, L., et al. (1992) Cell 71, 1093-1102;Townsend, S. E. and Allison, J. P. (1993) Science 259, 368-370; Baskar,S., et al. (1993) Proc. Natl. Acad. Sci. 90, 5687-5690). Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

[0162] In another aspect, a stimulatory form of one or more solublepeptides having an activity of a B lymphocyte antigen can beadministered to a tumor-bearing patient to provide a costimulatorysignal to T cells in order to induce anti-tumor immunity.

[0163] G. Administration of Therapeutic Forms of B Lymphocyte Antigens

[0164] The peptides of the invention are administered to subjects in abiologically compatible form suitable for pharmaceutical administrationin vivo to either enhance or suppress T cell mediated immune response.By “biologically compatible form suitable for administration in vivo” ismeant a form of the protein to be administered in which any toxiceffects are outweighed by the therapeutic effects of the protein. Theterm subject is intended to include living organisms in which an immuneresponse can be elicited, e.g., mammals. Examples of subjects includehumans, dogs, cats, mice, rats, and transgenic species thereof.Administration of a peptide having the activity of a novel B lymphocyteantigen as described herein can be in any pharmacological form includinga therapeutically active amount of peptide alone or in combination witha peptide having the activity of another B lymphocyte antigen and apharmaceutically acceptable carrier. Administration of a therapeuticallyactive amount of the therapeutic compositions of the present inventionis defined as an amount effective, at dosages and for periods of timenecessay to achieve the desired result. For example, a therapeuticallyactive amount of a peptide having B7-2 activity may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of peptide to elicit a desired response inthe individual. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0165] The active compound (e.g., peptide) may be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound may be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound.

[0166] To administer a peptide having B7-2 activity by other thanparenteral administration, it may be necessary to coat the peptide with,or co-administer the peptide with, a material to prevent itsinactivation. For example, a peptide hving B7-2 activity may beadministered to an individual in an appropriate carrier, diluent oradjuvant, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. Pharmaceutically acceptable diluents includesaline and aqueous buffer solutions. Adjuvant is used in its broadestsense and includes any immune stimulating compound such as interferon.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol 7:27).

[0167] The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

[0168] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, asorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0169] Sterile injectable solutions can be prepared by incorporatingactive compound (e.g., peptide having B7-2 activity) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient (e.g.,peptide) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0170] When the active compound is suitably protected, as describedabove, the protein may be orally administered, for example, with aninert diluent or an assimilable edible carrier. As used herein“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

[0171] It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0172] H. Identification of Cytokines Induced by Costimulation

[0173] The nucleic acid sequences encoding peptides having the activityof novel B lymphocyte antigens as described herein can be used toidentify cytokines which are produced by T cells in response tostimulation by a form of B lymphocyte antigen, e.g., B7-2. T cells canbe suboptimally stimulated in vitro with a primary activation signal,such as phorbol ester, anti-CD3 antibody or preferably antigen inassociation with an MHC class II molecule, and given a costimulatorysignal by a stimulatory form of B7-2 antigen, for instance by a celltransfected with nucleic acid encoding a peptide having B7-2 activityand expressing the peptide on its surface or by a soluble, stimulatoryform of the peptide. Known cytokines released into the media can beidentified by ELISA or by the ability of an antibody which blocks thecytokine to inhibit T cell proliferation or proliferation of other celltypes that is induced by the cytokine. An IL-4 ELISA kit is availablefrom Genzyme (Cambridge Mass.), as is an IL-7 blocking antibody.Blocking antibodies against IL-9 and IL-12 are available from GeneticsInstitute (Cambridge, Mass.).

[0174] An in vitro T cell costimulation assay as described above canalso be used in a method for identifying novel cytokines which may beinduced by costimulation. If a particular activity induced uponcostimulation, e.g., T cell proliferation, cannot be inhibited byaddition of blocking antibodies to known cytokines, the activity mayresult from the action of an unkown cytokine. Following costimulation,this cytokine could be purified from the media by conventional methodsand its activity measured by its ability to induce T cell proliferation.

[0175] To identify cytokines which prevent the induction of tolerance,an in vitro T cell costimulation assay as described above can be used.In this case, T cells would be given the primary activation signal andcontacted with a selected cytokine, but would not be given thecostimulatory signal. After washing and resting the T cells, the cellswould be rechallenged with both a primary activation signal and acostimulatory signal. If the T cells do not respond (e.g., proliferateor produce IL-2) they have become tolerized and the cytokine has notprevented the induction of tolerance. However, if the T cells respond,induction of tolerance has been prevented by the cytokine. Thosecytokines which are capable of preventing the induction of tolerance canbe targeted for blockage in vivo in conjunction with reagents whichblock B lymphocyte antigens as a more efficient means to inducetolerance in transplant recipients or subjects with autoimmune diseases.For example, one could administer a B7-2 blocking reagent together witha cytokine blocking antibody to a subject.

[0176] I. Identification of Molecules which Inhibit Costimulation

[0177] Another application of the peptide having the activity of a novelB lymphocyte antigen of the invention (e.g., B7-2 and B7-3) is the useof one or more of these peptides in screening assays to discover as yetundefined molecules which are inhibitors of costimulatory ligand bindingand/or of intracellular signaling through T cells followingcostimulation. For example, a solid-phase binding assay using a peptidehaving the activity of a B lymphocyte antigen, such as B7-2, could beused to identify molecules which inhibit binding of the antigen with theappropriate T cell ligand (e.g., CTLA4, CD28). In addition, an in vitroT cell costimulation assay as described above could be used to identifymolecules which interfere with intracellular signaling through the Tcells following costimulation as determined by the ability of thesemolecules to inhibit T cell proliferation and/or cytokine production(yet which do not prevent binding of B lymphocyte antigens to theirreceptors). For example, the compound cyclosporine A inhibits T cellactivation through stimulation via the T cell receptor pathway but notvia the CD28/CTLA4 pathway. Therefore, a different intracellularsignaling pathway is involved in costimulation. Molecules whichinterfere with intracellular signaling via the CD28/CTLA4 pathway may beeffective as immunosuppressive agents in vivo (similar to the effects ofcyclosporine A).

[0178] J. Identification of Molecules which Modulate B LymphocyteAntigen Expression

[0179] The monoclonal antibodies produced using the proteins andpeptides of the current invention can be used in a screening assay formolecules which modulate the expression of B lymphocyte antigens oncells. For example, molecules which effect intracellular signaling whichleads to induction of B lymphocyte antigens, e.g. B7-2 or B7-3, can beidentified by assaying expression of one or more B lymphocyte antigenson the cell surface. Reduced immunofluorescent staining by an anti-B7-2antibody in the presence of the molecule would indicate that themolecule inhibits intracellular signals. Molecules which upregulate Blymphocyte antigen expression result in an increased immunofluorescentstaining. Alternatively, the effect of a molecule on expression of a Blymphocyte antigen, such as B7-2, can be determined by detectingcellular B7-2 mRNA levels using a B7-2 cDNA as a probe. For example, acell which expresses a peptide having B7-2 activity can be contactedwith a molecule to be tested, and an increase or decrease in B7-2 mRNAlevels in the cell detected by standard technique, such as Northernhybridization analysis or conventional dot blot of mRNA or totalpoly(A⁺)RNAs using a B7-2 cDNA probe labeled with a detectable marker.Molecules which modulate B lymphocyte antigen expression may be usefulltherapeutically for either upregulating or downregulating immuneresponses alone or in conjunction with soluble blocking or stimulatingreagents. For instance, a molecule which inhibits expression of B7-2could be administered together with a B7-2 blocking reagent forimmunosuppressive purposes. Molecules which can be tested in theabove-described assays include cytokines such as IL-4, γINF, IL-10,IL-12, GM-CSF and prostagladins.

[0180] This invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences and published patent applications cited throughout thisapplication are hereby incorporated by reference.

[0181] The following methodology was used in Examples 1, 2 and 3.

[0182] Methods and Materials

[0183] A. Cells

[0184] Mononuclear cells were isolated by Ficoll-Hypaque densitygradient centrifugation from single cell suspensions of normal humanspleens and were separated into E− and E+ fractions by resetting withsheep red blood cells (Boyd, A. W., et al. (1985) J. Immunol. 134,1516). B cells were purified from the E− fraction by adherence ofmonocytes on plastic and depletion of residual T, natural killer cells(NK) and residual monocytes by two treatments with anti-MsIgG andanti-MsIgM coated magnetic beads (Advanced Magnetics, Cambridge, Mass.),using monoclonal antibodies: anti-CD4, -CD8, -CD11b, -CD14 and -CD16.CD4+ T cells were isolated from the E+ fraction of the same spleensafter adherence on plastic and depletion of NK, B cells and residualmonocytes with magnetic beads and monoclonal antibodies: anti-CD20,-CD11b, -CD8 and -CD16. CD28+ T cells were identically isolated from theE+ fraction using anti-CD20, -CD11b, -CD 14 and -CD16 monoclonalantibodies. The efficiency of the purification was analyzed by indirectimmunofluorescence and flow cytometry using an EPICS flow cytometer(Coulter). B cell preparations were >95% CD20+, <2% CD3+, <1% CD14+.CD4+ T cell preparations were >98% CD3+, >98% CD4+, <1% CD8+, <1% CD20+,<1% CD14+. CD28+ T cell preparations were >98% CD3+, >98% CD28+, <1%CD20+, <1% CD14+.

[0185] B. Monoclonal Antibodies and Fusion Proteins

[0186] Monoclonal antibodies were used as purified Ig unless indicatedotherwise: anti-B7:133, IgM is a blocking antibody and has beenpreviously described (Freedman, A. S. et al. (1987) Immunol. 137,3260-3267); anti-B7:B1.1, IgG1 (RepliGen Corp., Cambridge, Mass.)(Nickoloff, B., et al (1993) Am. J. Pathol. 142, 1029-1040) is anon-blocking monoclonal antibody; BB-1: IgM is a blocking antibody (Dr.E. Clark, University of Washington, Seattle, Wash.) (Yokochi, T., et al.(1982) J. Immunol. 128, 823-827); anti-CD20: B1, IgG2a (Stashenko, P.,et al.(1980) J. Immunol. 125, 1678-1685); anti-B5: IgM (Freedman, A., etal. (1985) J. Immunol. 134, 2228-2235); anti-CD8: 7PT 3F9, IgG2a;anti-CD4: 19Thy5D7, IgG2a; anti-CD11b: Mol, IgM and anti-CD14: Mo2, IgM(Todd, R, et al. (1981) J. Immunol. 126, 1435-1442); anti-MHC class II:9-49, IgG2a (Dr R. Todd, University of Michigan, Ann Arbor) (Todd, R.I., et al. (1984) Hum Immunol. 10, 23-40; anti-CD28: 9.3, IgG2a (Dr. C.June, Naval Research Institute, Bethesda) (Hansen, J. A., et al. (1980)Immunogenetics. 10, 247-260); anti-CD16: 3G8, IgG1 (used as ascites)(Dr. J. Ritz, Dana-Farber Cancer Institute, Boston); anti-CD3: OKT3,IgG2a hybridoma was obtained from the American Type Culture Collectionand the purified monoclonal antibody was adhered on plastic plates at aconcentration of 1 μg/ml; anti-CD28 Fab fragments were generated fromthe 9.3 monoclonal antibody, by papain digestion and purification on aprotein A column, according to the manufacturer's instructions (Pierce,Rockford, Ill.). Human CTLA4 fusion protein (CTLA4Ig) and controlfuision protein (control-Ig) were prepared as previously described(Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci USA 90:6586-6590);Boussiotis, V., et al J. Exp. Med. (accepted for publication)).

[0187] C. CHO Cell Transfection

[0188] B7-1 transfectants (CHO-B7) were prepared from the B7-1 negativechinese hamster ovary (CHO) cell line, fixed with paraformaldehyde andused as previously described (Gimmi, C. D., et al. Proc. Natl. Acad. SciUSA 88, 6575-6579).

[0189] D. In Vitro B Cell Activation and Selection of B7+ and B7− Cells

[0190] Splenic B cells were cultured at 2×10⁶ cells/ml in completeculture media, {RPMI 1640 with 10% heat inactivated fetal calf serum(FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml),streptomycin sulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, intissue culture flasks and were activated by crosslinking of sIg withaffinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads(Bio-Rad), Richmond, Calif.) (Boyd, A. W., et al., (1985) J. Immunol.134,1516) or by crosslinking of MHC class II with 9-49 antibody coupledto Affi-Gel 702 beads. B cells activated for 72 hours, were used astotal activated B cell populations or were indirectly stained withanti-B7 (B1.1) monoclonal antibody and fluorscein isothiocyanate (FITC)labeled goat anti-mouse immunoglobulin (Fisher, Pittsburgh, Pa.), andfractionated into B7-1+ and B7-1− populations by flow cytometric cellsorting (EPICS Elite flow cytometer, Coulter).

[0191] E. Immunoflouorescence and Flow Cytometry

[0192] For surface phenotype analysis populations of B cells activatedby either sIg or MHC class II crosslinking for 6, 12, 24, 48, 72 and 96hours were stained with either anti-B7 (133), BB-1 monoclonalantibodies, control IgM antibody, CTLA4Ig or control-Ig. Cellsuspensions were stained by two step indirect membrane staining with 10μg/ml of primary monoclonal antibody followed by the appropriatesecondary reagents. Specifically, immunoreactivity with anti-B7 (133)and BB-1 monoclonal antibodies was studied by indirect staining usinggoat anti-mouse Ig or immunoglobulin FITC (Fisher) as secondary reagentand immunoreactivity with fusion proteins was studied using biotinylatedCTLA4Ig or biotinylated control-Ig and streptavidin-phycoerythrin assecondary reagent. PBS containing 10% AB serum was used as diluent andwash media. Cells were fixed with 0.1% paraformaldehyde and analyzed ona flow cytometer (EPICS Elite Coulter).

[0193] F. Proliferation Assay

[0194] T cells were cultured at a concentration of 1×10⁵ cells per wellin 96-well flat bottom microtiter plate at 37° C. for 3 days in 5% CO₂.Syngeneic activated B cells (total B cell population or B7+ and B7−fractions) were irradiated (2500 rad) and added into the cultures at aconcentration of 1×10⁵ cells per well. Factors under study were added tothe required concentration for a total final volume of 200 μl per well.When indicated, T cells were incubated with anti-CD28 Fab (finalconcentration of 10 μg/ml), for 30 minutes at 4° C., prior to additionin experimental plates. Similarly, CHO-B7 or B cells were incubated withCTLA4Ig or control-Ig (10 μg/ml) for 30 minutes at 4° C. Thymidineincorporation as an index of mitogenic activity, was assessed afterincubation with 1 μCi (37kBq) of {methyl-³H} thymidine (Du Pont, Boston,Mass.) for the last 15 hours of the culture. The cells were harvestedonto filters and the radioactivity on the dried filters was measured ina Pharmacia beta plate liquid scintilation counter.

[0195] G. IL-2 and IL-4 Assay

[0196] IL-2 and IL-4 concentrations were assayed by ELISA (R&D Systems,Minneapolis, Minn. and BioSource, Camarillo, Calif.) in culturesupernatants collected at 24 hours after initiation of the culture.

EXAMPLE 1 Expression of a Novel CTLA4 Ligand on Activated B Cells whichInduces T Cell Proliferation

[0197] Since crosslinking surface Ig induces human resting B cells toexpress B7-1 maximally (50-80%) at 72 hours, the ability of activatedhuman B lymphocytes to induce submitogenically activated T cells toproliferate and secrete IL-2 was determined. FIG. 1 depicts thecostimulatory response of human splenic CD28+ T cells, submitogenicallyactivated with anti-CD3 monoclonal antibody, to either B7 (B7-1)transfected CHO cells (CHO-B7) or syngeneic splenic B cells activatedwith anti-Ig for 72 hours. ³H-Thymidine incorporation was assessed forthe last 15 hours of a 72 hours culture. IL-2 was assessed by ELISA insupernatants after 24 hours of culture (Detection limits of the assay:31-2000 pg/ml). FIG. 1 is representative of seventeen experiments.

[0198] Submitogenically activated CD28+ T cells proliferated andsecreted high levels of IL-2 in response to B7-1 costimulation providedby CHO-B7 (FIG. 1, panel a). Both proliferation and IL-2 secretion weretotally inhibited by blocking the B7-1 molecule on CHO cells with eitheranti-B7-1 monoclonal antibody or by a fusion protein for its highaffinity receptor, CTLA4. Similarly, proliferation and IL-2 secretionwere abrogated by blocking B7-1 signalling via CD28 with Fab anti-CD28monoclonal antibody. Control monoclonal antibody or control fusionprotein had no effect. Nearly identical costimulation of proliferationand IL-2 secretion was provided by splenic B cells activated withanti-Ig for 72 hours (panel b). Though anti-B7-1 monoclonal antibodycould completely abrogate both proliferation and IL-2 secretiondelivered by CHO-B7, anti-B7-1 monoclonal antibody consistentlyinhibited proliferation induced by activated B cells by only 50% whereasIL-2 secretion was totally inhibited. In contrast to the partialblockage of proliferation induced by anti-B7-1 monoclonal antibody, bothCTLA4Ig and Fab anti-CD28 monoclonal antibody completely blockedproliferation and IL-2 secretion. These results are consistent with thehypothesis that activated human B cells express one or more additionalCTLA4/CD28 ligands which can induce T cell proliferation and IL-2secretion.

EXAMPLE 2 Activated Human Splenic B Cells Express CTLA4 Ligand(s)Distinct from B7-1

[0199] In light of the above observations, whether other CTLA4 bindingcounter-receptors were expressed on activated B cells was determined. Tothis end, human splenic B cells were activated for 72 hours with anti-Igand then stained with an anti-B7-1 monoclonal antibody (B1.1) which doesnot inhibit B7-1 mediated costimulation. Fluoroscein isothiocyanate(FITC) and mAb B1.1 were used with flow cytometric cell sorting toisolate B7-1⁺ and B7-1⁻ fractions. The resulting post-sort positivepopulation was 99% B7-1⁺ and the post-sort negative population was 98%B7-1⁻ (FIG. 2).

[0200] To examine the costimulatory potential of each population, humansplenic CD28+ T cells were submitogenically stimulated with anti-CD3monoclonal antibody in the presence of irradiated B7-1+ or B7-1− anti-Igactivated (72 hours) splenic B cells. ³H-Thymidine incorporation wasassessed for the last 15 hours of a 72 hours culture. IL-2 was assessedby ELISA in supernatants after 24 hours of culture (Detection limits ofthe assay: 31-2000 pg/ml). The results of FIG. 3 are representative often experiments. B7-1+ B cells induced anti-CD3 activated T cells toproliferate and secrete IL-2 (FIG. 3a) but not IL-4. As was observedwith the unfractionated activated B cell population, anti-B7-1monoclonal antibody (133) inhibited proliferation only 50% butconsistently abrogated IL-2 secretion. As above, CTLA4Ig binding orblockade of CD28 with Fab anti-CD28 monoclonal antibody completelyinhibited both proliferation and IL-2 secretion. Control monoclonalantibody and control-Ig were not inhibitory. In an attempt to identifyother potential CTLA4/CD28 binding costimulatory ligand(s) which mightaccount for the residual, non-B7 mediated proliferation delivered by B7+B cells, the effect of BB-1 monoclonal antibody on proliferation andIL-2 secretion was examined. As seen, BB-1 monoclonal antibodycompletely inhibited both proliferation and IL-2 secretion (FIG. 3a).FIG. 3b displays the costimulatory potential of B7-1− activated humansplenic B cells. Irradiated B7-1− activated (72 hr) B cells could alsodeliver a significant costimulatory signal to submitogenically activatedCD4+ lymphocytes. This costimulation was not accompanied by detectableIL-2 (FIG. 3b) or IL-4 accumulation and anti-B7-1 monoclonal antibodydid not inhibit proliferation. However, CTLA4Ig, Fab anti-CD28monoclonal antibody, and BB-1 monoclonal antibody all completelyinhibited proliferation.

[0201] Phenotypic analysis of the B7-1+ and B7-1− activated splenic Bcells confirmed the above functional results. FIG. 4 shows the cellsurface expression of B7-1, B7-2 and B7-3 on fractionated B7-1⁺ andB7-1⁻ activated B cell. As seen in FIG. 4, B7-1+ activated splenic Bcells stained with anti-B7-1 (133) monoclonal antibody, BB-1 monoclonalantibody, and bound CTLA4-Ig. In contrast, B7− activated splenic B cellsdid not stain with anti-B7-1 (133) monoclonal antibody but did stainwith BB-1 monoclonal antibody and CTLA4Ig. These phenotypic andfunctional results demonstrate that both B7-1+ and B7-1− activated (72hours) human B lymphocytes express CTLA4 binding counter-receptor(s)which: 1) can induce submitogenically activated T cells to proliferatewithout detectable IL-2 secretion; and 2) are identified by the BB-1monoclonal antibody but not anti-B7-1 monoclonal antibody. Thus, theseCTLA4/CD28 ligands can be distinguished on the basis of their temporalexpression after B cell activation and their reactivity with CTLA4Ig andanti-B7 monoclonal antibodies. The results of FIG. 4 are representativeof five experiments.

EXAMPLE 3 Three Distinct CTLA4/CD28 Ligands are Expressed FollowingHuman B Cell Activation

[0202] To determine the sequential expression of CTLA4 bindingcounter-receptors following activation, human splenic B cells wereactivated by crosslinking of either surface Ig or MHC class II and theexpression of B7-1, B7-3 and B7-2 binding proteins were examined by flowcytometric analysis. Ig or MHC class II crosslinking induced a similarpattern of CTLA4Ig binding (FIGS. 5 and 6). FIG. 5 is representative ofthe results of 25 experiments for anti-B7-1 and BB-1 binding and 5experiments for CTLA4Ig binding. FIG. 6 is representative of 25experiments for anti-B7-1 binding and 5 experiments for CTLA4Ig binding.The results of these experiments indictes that prior to 24 hours, noneof these molecules are expressed. At 24 hours post-activation, themajority of cells express a protein that binds CTLA4Ig (B7-2), however,fewer than 20% express either B7-1 or B7-3. Crosslinking of MHC class IIinduces maximal expression and intensity of B7-1 and B7-3 at 48 hourswhereas crosslinking of Ig induces maximal expression at 72 hours andexpression declines thereafter. These results suggest that an additionalCTLA4 binding counter-receptor is expressed by 24 hours and that thetemporal expression of the distinct B7-1 and B7-3 proteins appears tocoincide.

[0203] A series of experiments was conducted to determine whether thetemporal expression of CTLA4 binding counter-receptors differentiallycorrelated with their ability to costimulate T cell proliferation and/orIL-2 secretion. Human splenic CD28+ T cells submitogenically stimulatedwith anti-CD3 were cultured for 72 hours in the presence of irradiatedhuman splenic B cells that had been previously activated in vitro by sIgcrosslinking for 24, 48, or 72 hours. IL-2 secretion was assessed byELISA in supernatants after 24 hours and T cell proliferation asassessed by ³H-thymidine incorporation for the last 15 hours of a 72hour culture. The results of FIG. 7 are representative of 5 experiments.As seen in FIG. 7a, 24 hour activated B cells provided a costimulatorysignal which was accompanied by modest levels of IL-2 production,although the magnitude of proliferation was significantly less thanobserved with 48 and 72 hours activated human B cells (note differencesin scale for ³H-Thymidine incorporation). Neither proliferation nor IL-2accumulation was inhibited by anti-B7-1 (133) or BB-1. In contrast, withCTLA4Ig and anti-CD28 Fab monoclonal antibody totally abrogatedproliferation and IL-2 accumulation. B cells activated for 48 hours,provided costimulation which resulted in nearly maximal proliferationand IL-2 secretion (FIG. 7b). Here, anti-B7-1 (133) monoclonal antibody,inhibited proliferation approximately 50% but totally blocked IL-2accumulation. BB-1 monoclonal antibody totally inhibited bothproliferation and IL-2 secretion. As above, CTLA4Ig and Fab anti-CD28also totally blocked proliferation and IL-2 production. Finally, 72 houractivated B cells induced T cell response identical to that induced by48 hour activated B cells. Similar results are observed if thesubmitogenic signal is delivered by phorbol myristic acid (PMA) and ifthe human splenic B cells are activated by MHC class II rather than Igcrosslinking. These results indicate that there are three CTLA4 bindingmolecules that are temporarily expressed on activated B cells and eachcan induce submitogenically stimulated T cells to proliferate. Two ofthese molecules, the early CTLA4 binding counter-receptor (B7-2) andB7-1 (133) induce IL-2 production whereas B7-3 induces proliferationwithout detectable IL-2 production.

[0204] Previous studies provided conflicting evidence whether theanti-B7 monoclonal antibody, 133 and monoclonal antibody BB-1 identifiedthe same molecule (Freedman, A. S. et al. (1987) Immunol. 137,3260-3267; Yokochi, T., et al. (1982) J. Immunol. 128, 823-827; Freeman,G. J., et al. (1989) J. Immunol. 143, 2714-2722.). Although bothmonoclonal antibodies identified molecules expressed 48 hours followinghuman B-cell activation, several reports suggested that B7 (B7-1) andthe molecule identified by monoclonal antibody BB-1 were distinct sincethey were differentially expressed on cell lines and B cell neoplasms(Freedman, A. S. et al. (1987) Immunol. 137, 3260-3267; Yokochi, T., etal. (1982) J. Immunol. 128, 823-827; Freeman, G. J., et al. (1989) J.Immunol. 143, 2714-2722; Clark, E and Yokochi, T. (1984) LeukocyteTyping, 1st International References Workshop. 339-346; Clark, E., etal. (1984) Leukocyte Typing, 1st International References Workshop.740). In addition, immunoprecipitation and Western Blotting with theseIgM monoclonal antibodies suggested that they identified differentmolecules (Clark, E and Yokochi, T. (1984) Leukocyte Typing, 1stInternational References Workshop. 339-346; Clark, E., et al. (1984)Leukocyte Typing, 1st International References Workshop. 740). Theoriginal anti-B7 monoclonal antibody, 133, was generated by immunizationwith anti-immunoglobulin activated human B lymphocytes whereas the BB-1monoclonal antibody was generated by immunization with a baboon cellline. Thus, the BB-1 monoclonal antibody must identify an epitope onhuman cells that is conserved between baboons and humans.

[0205] Following the molecular cloning and expression of the human B7gene (B7-1), B7 transfected COS cells were found to be identicallystained with the anti-B7 (133) and BB-1 monoclonal antibodies and thatthey both precipitated the identical broad molecular band (44-54 kD)strongly suggesting that they identified the same molecule (Freeman, G.J., et al. (1989) J. Immunol. 143, 2714-2722). This observation wasunexpected since the gene encoding the molecule identified by the BB-1monoclonal antibody had been previously mapped to chromosome 12 (Katz,F. E., et al. (1985) Eur. J. Immunol. 103-6), whereas the B7 gene waslocated by two groups on chromosome 3 (Freeman, G. J., et al. (1992)Blood. 79, 489-494; Selvakumar, A., et al. (1992) Immunogenetics 36,175-181.). Subsequently, additional discrepancies between the phenotypicexpression of B7 (B7-1) and the molecule identified by the BB-1monclonal antibody were noted. BB-1 monoclonal antibody stained thymicepithelial cells (Turka, L. A., et al. (1991) J. Immunol. 146, 1428-36;Munro, J. M., et al. Blood submitted.) and keratinocytes (Nickoloff, B.,et al (1993) Am. J. Pathol. 142, 1029-1040; Augustin, M., et al. (1993)J. Invest. Dermatol. 100, 275-281.) whereas anti-B7 did not. Recently,Nickoloffet al. (1993) Am. J. Pathol. 142, 1029-1040, reporteddiscordant expression of the molecule identified by the BB-1 monoclonalantibody and B7 on keratinocytes using a BB-1 and anti-B7 (B1.1 and 133)monoclonal antibodies. Nickoloff et al. also demonstrated that theseBB-1 positive cells did not express B7 mRNA yet bound CD28 transfectedCOS cells providing further support for the existence of a distinctprotein which binds monoclonal antibody BB-1.

[0206] The present findings confirm that there is an additional CTLA4counter-receptor identified by the BB-1 monoclonal antibody, B7-3, andthat this protein appears to be function ally distinct from B7-1 (133).Although the expression of B7-1 and B7-3 following B cell activationappears to be concordant on B7 positive B cells, these studiesdemonstrate that the B7-3 molecule is also expressed on B7 negativeactivated B cells. More importantly, the B7-3 molecule appears to becapable of inducing T cell proliferation without detectable IL-2 or IL-4production. This result is similar to the previous observation thatICAM-1 could costimulate T cell proliferation without detectable IL-2 orIL-4 production (Boussiotis, V., et al J. Exp. Med. (accepted forpublication)). These data indicate that the BB-1 monoclonal antibodyrecognizes an epitope on the B7-1 protein and that this epitope is alsofound on a distinct B7-3 protein, which also has costimulatory function.Phenotypic and blocking studies demonstrate that the BB-1 monoclonalantibody could detect one (on B7 negative cells) or both (on B7 positivecells) of these proteins. In contrast, the anti-B7 monoclonalantibodies, 133 and B1.1 detect only the B7-1 protein. Taken together,these results suggest that by 48 hours post B-cell activation bycrosslinking of surface immunoglobulin or MHC class II, B cells expressat least two distinct CTLA4 binding counter-receptors, one identified byboth anti-B7 and BB-1 monclonal antibodies and the other identified onlyby BB-1 monoclonal antibody.

[0207] The B7-2 antigen is not detectable on activated B cells after 12hours, but by 24 hours it is strongly expressed and functional. Thismolecule appears to signal via CD28 since proliferation and IL-2production are completely blocked by Fab anti-CD28 monoclonal antibody.At 48 hours post activation, IL-2 secretion seems to be accounted for byB7-1 costimulation, since anti-B7 monoclonal antibody completelyinhibits IL-2 production.

[0208] Previous studies and results presented here demonstrate that B7(B7-1) is neither expressed (Freedman, A. S. et al. (1987) Immunol. 137,3260-3267; Freedman, A. S., et al. (1991) Cell. Immunol. 137, 429-437)nor capable of costimulating T cell proliferation or IL-2 secretionuntil 48 hours post B-cell activation. Previous studies have shown thatactivation of T cells via the TCR in the absence of costimulation(Gimmi, C. D., et al. (1993) Proc. Natl. Acad. Sci USA 90:6586-6590;Schwartz, R. H., et al. (1989) Cold Spring Harb. Symp. Quant. Biol 54,605-10; Beverly, B., et al. (1992) Int. Immunol. 4, 661-671.) and lackof IL-2 (Boussiotis, V., et al J. Exp. Med. (submitted); Beverly, B., etal. (1992) Int. Immunol. 4, 661-671; Wood, M., et al. (1993) J. Exp.Med. 177, 597-603) results in anergy. If B7-1 were the onlycostimulatory molecule capable of inducing IL-2 secretion, T cells wouldbe anergized within the first 24 hours following activation since thereis no B7-1 present to costimulate IL-2 production. Therefore, theexistence of another, early inducible costimulatory molecule, which cancostimulate IL-2 secretion during the first 24 hours would be necessaryto induce an effective immune response rather than anergy. Theappearance of the early CTLA4 binding counter-receptor, B7-2, between 12and 24 hours post B cell activation, fulfills this function.

[0209] Two observations shed light on the biologic and potentialclinical significance of these two additional CTLA4 bindingcounter-receptors. First, B7 (B7-1) deficient mouse has been developedand its antigen presenting cells were found to still bind CTLA4Ig(Freeman and Sharpe manuscript in preparation). This mouse is viable andisolated mononuclear cells induce detectable levels of IL-2 whencultured with T cells in vitro. Therefore, an alternative CD28costimulatory counter-receptor or an alternative IL-2 producing pathwaymust be function al. Second, thus far the most effective reagents toinduce antigen specific anergy in murine and human systems are CTLA4Igand Fab anti-CD28, whereas anti-B7 monoclonal antibodies have been muchless effective (Harding, F. A., et al. (1992) Nature. 356, 607-609;Lenschow, D. J., et al. (1992) Science. 257, 789-792; Chen, L., et al.(1992) Cell. 71, 1093-1102; Tan, P., et al. (1993) J. Exp. Med. 177,165-173.). These observations are also consistent with the hypothesisthat alternative CTLA4/CD28 ligands capable of inducing IL-2 exist, andtaken together with the results presented herein, suggest that all threeCTLA4 binding counter-receptors may be critical for the induction of Tcell immunity. Furthermore, their blockade will likely be required forthe induction of T cell anergy. Identical results have been observed inthe murine system with the identification of two CTLA4 binding ligands,corresponding to the human B7-1 and B7-2 molecules. APCs in the B7deficient mouse bind to the CTLA4 and can induce IL-2 secretion. Takentogether, these observations indicate that multiple CTLA-4 bindingcounter-receptors exist and sequentially costimulate T cell activationin the murine system.

EXAMPLE 4 Cloning, Sequencing and Expression of the B7-2 Antigen

[0210] A. Construction of cDNA Library

[0211] A cDNA library was constructed in the pCDM8 vector (Seed, Nature,329:840 (1987)) using poly(A)⁺ RNA from the human anti-IgM activated Bcells as described (Aruffo et al, Proc. Natl. Acad. Sci. USA, 84:3365(1987)). Splenic B cells were cultured at 2×10⁶ cells/ml in completeculture media, {RPMI 1640 with 10% heat inactivated fetal calf serum(FCS), 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 units/ml),streptomycin sulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, intissue culture flasks and were activated by crosslinking of sIg withaffinity purified rabbit anti-human IgM coupled to Affi-Gel 702 beads(Bio-Rad), Richmond, Calif.) (Boyd, A. W., et al., (1985) J. Immunol.134,1516). Activated B cells were harvested after ⅙, ½, 4, 8 12, 24, 48,72 and 96 hours.

[0212] RNA was prepared by homogenizing activated B cells in a solutionof 4M guanidine thiocyanate, 0.5% sarkosyl, 25 mM EDTA, pH 7.5, 0.13%Sigma anti-foam A, and 0.7% mercaptoethanol. RNA was purified from thehomogenate by centrifugation for 24 hour at 32,000 rpm through asolution of 5.7M CsCl, 10 mM EDTA, 25 mM Na acetate, pH 7. The pellet ofRNA was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 andextracted with two volumes of 50% phenol, 49% chloroform, 1% isoamylalcohol. RNA was ethanol precipitated twice. Poly(A)⁺ RNA used in cDNAlibrary construction was purified by two cycles of oligo (dT)-celluloseselection.

[0213] Complementary DNA was synthesized from 5.5 μg of anti-IgMactivated human B cell poly(A)⁺ RNA in a reaction containing 50 mM Tris,pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 500 μM dATP, dCTP,dGTP, dTTP, 50 μg/ml oligo(dT)₁₂₋₁₈, 180 units/ml RNasin, and 10,000units/ml Moloney-MLV reverse transcriptase in a total volume of 55 μl at37° for 1 hr. Following reverse transcription, the cDNA was converted todouble-stranded DNA by adjusting the solution to 25 mM Tris, pH 8.3, 100mM KCl, 5 mM MgCl₂, 250 μM each dATP, dCTP, dGTP, dTTP, 5 mMdithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/ml ribonucleaseH and incubating at 16 for 2 hr. EDTA was added to 18 mM and thesolution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5M ammonium acetate and with 4 microgramsof linear polyacrylamide as carrier. In addition, cDNA was synthesizedfrom 4 μg of anti-IgM activated human B cell poly(A)⁺ RNA in a reactioncontaining 50 mM Tris, pH 8.8, 50 μg/ml oligo(dT)₁₂₋₁₈, 327 units/mlRNasin, and 952 units/ml AMV reverse transcriptase in a total volume of100 μl at 42° for 0.67 hr. Following reverse transcription, the reversetranscriptase was inactivated by heating at 70° for 10 min. The cDNA wasconverted to double-stranded DNA by adding 320 μl H₂O and 80 μl of asolution of 0.1M Tris, pH 7.5, 25 mM MgCl₂, 0.5M KCl, 250 μg/ml bovineserum albumin, and 50 mM dithiothreitol, and adjusting the solution to200 μM each dATP, dCTP, dGTP, dTTP, 50 units/ml DNA polymerase I, 8units/ml ribonuclease H and incubating at 16° C. for 2 hours. EDTA wasadded to 18 mM and the solution was extracted with an equal volume of50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA was precipitatedwith two volumes of ethanol in the presence of 2.5M ammonium acetate andwith 4 micrograms of linear polyacrylamide as carrier.

[0214] The DNA from 4 μg of AMV reverse transcription and 2 μg ofMoloney MLV reverse transcription was combined. Non-selfcomplementaryBstXI adaptors were added to the DNA as follows: The double-strandedcDNA from 6 μg of poly(A)⁺ RNA was incubated with 3.6 μg of a kinasedoligonucleotide of the sequence CTTTAGAGCACA (SEQ ID NO:15) and 2.4 μgof a kinased oligonucleotide of the sequence CTCTAAAG (SEQ ID NO:16) ina solution containing 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350μg/ml bovine serum albumin, 7 mM mercaptoethanol, 1.0 mM ATP, 2 mMdithiothreitol, 1 mM spermidine, and 600 units T4 DNA ligase in a totalvolume of 0.45 ml at 15° C. for 16 hours. EDTA was added to 34 mM andthe solution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5M ammonium acetate.

[0215] DNA larger than 600 bp was selected as follows: The adaptored DNAwas redissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1%sarkosyl and chromatographed on a Sepharose CL-4B column in the samebuffer. DNA in the void volume of the column (containing DNA greaterthan 600 bp) was pooled and ethanol precipitated.

[0216] The pCDM8 vector was prepared for cDNA cloning by digestion withBstXI and purification on an agarose gel. Adaptored DNA from 6 μg ofpoly(A)⁺ RNA was ligated to 2.25 μg of BstXI cut pCDM8 in a solutioncontaining 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovineserum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1mM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5 mlat 15° for 24 hr. The ligation reaction mixture was transformed intocompetent E.coli MC1061/P3 and a total of 4,290,000 independent cDNAclones were obtained.

[0217] Plasmid DNA was prepared from a 500 ml culture of the originaltransformation of the cDNA library. Plasmid DNA was purified by thealkaline lysis procedure followed by twice banding in CsCl equilibriumgradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1987)).

[0218] B. Cloning Procedure

[0219] In the first round of screening, thirty 100 mm dishes of 50%confluent COS cells were transfected with 0.05 μg/ml anti-IgM activatedhuman B cells library DNA using the DEAE-Dextran method (Seed et al,Proc. Natl. Acad. Sci. USA, 84:3365 (1987)). The cells were trypsinizedand re-plated after 24 hours. After 47 hours, the cells were detached byincubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37° C. for 30min. The detached cells were treated with 10 μg/ml/CTLA4Ig and CD28Igfor 45 minutes at 4° C. Cells were washed and distributed into panningdishes coated with affinity-purified Goat anti-human IgG antibody andallowed to attach at room temperature. After 3 hours, the plates weregently washed twice with PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide, 5% FCSand once with 0.1 5M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS. Episomal DNAwas recovered from the panned cells and transformed into E. coliDH10B/P3. The plasmid DNA was re-introduced into COS cells viaspheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci USA,84:3365 (1987)) and the cycle of expression and panning was repeatedtwice. In the second and third rounds of selection, after 47 hours, thedetached COS cells were first incubated with α-B7-1 mAbs (133 and B1.1,10 μg/ml), and COS cells expressing B7-1 were removed by α-mouse IgG andIgM coated magnetic beads. COS cells were then treated with 10 μg/ml ofhuman CTLA4Ig (hCTLA4Ig) and human CD28Ig (hCD28Ig) and human B7-2expressing COS cells were selected by panning on dishes with goatanti-human IgG antibody plates. After the third round, plasmid DNA wasprepared from individual colonies and transfected into COS cells by theDEAE-Dextran method. Expression of B7-2 on transfected COS cells wasanalyzed by indirect immunofluorescence with CTLA4Ig.

[0220] After the final round of selection, plasmid DNA was prepared fromindividual colonies. A total of 4 of 48 candidate clones contained acDNA insert of approximately 1.2 kb. Plasmid DNA from these four cloneswas transfected into COS cells. All four clones were strongly positivefor B7-2 expression by indirect immunofluorescence using CTLA4Ig andflow cytometric analysis.

[0221] C. Sequencing

[0222] The B7-2 cDNA insert in clone29 was sequenced in the pCDM8expression vector employing the following strategy. Initial sequencingwas performed using sequencing primers T7, CDM8R (Invitrogen) homologousto pCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see TableI). Sequencing was performed using dye terminator chemistry and an ABIautomated DNA sequencer. (ABI, Foster City, Calif.). DNA sequenceobtained using these primers was used to design additional sequencingprimers (see Table I). This cycle of sequencing and selection ofadditional primers was continued until the B7-2 cDNA was completelysequenced on both strands. TABLE I T7(F) (SEQ ID NO:3)5′d[TAATACGACTCACTATAGGG]3′ CDM8(R) (SEQ ID NO:4)5′d[TAAGGTTCCTTCACAAAG]3′ CDM8 RGV(2) 5′d[ACTGGTAGGTATGGAAGATCC]3′ (SEQID NO:5) HBX29-5P(2R) 5′d[ATGCGAATCATTCCTGTGGGC]3′ (SEQ ID NO:6)HBX29-5P(2F) 5′d[AAAGCCCACAGGAATGATTCG]3′ (SEQ ID NO:7) HBX29-5P5′d[CTCTCAAAACCAAABCCTGAG]3′ (SEQ ID NO:8) 5PA (SEQ ID NO:9)5′d[TTAGGTCACAGCAGAAGCAGC]3′ 5PA (3FA) 5′d[TCTGGAAACTGACAAGACGCG]3′ (SEQID NO:10) HBX29-5P(1R) 5′d[CTCAGGCTTTGGTTTTGAGAG]3′ (SEQ ID NO:11)HBX29-3P(1R) 5′d[CACTCTCTTCCCTCTCCATTG]3′ (SEQ ID NO:12) HBX29-5P(3R)5′d[GACAAGCTGATGGAAACGTCG]3′ (SEQ ID NO:13) HBX29-3P(1P)5′d[CAATGGAGAGGGAAGAGAGTG]3′ (SEQ ID NO:14)

[0223] The human B7-2 clone 29 contained an insert of 1,120 base pairswith a single long open reading frame of 987 nucleotides andapproximately 27 nucleotides of 3′ noncoding sequences (FIG. 8 (SEQ IDNO:1)). The predicted amino acid sequence encoded by the open readingframe of the protein is shown below the nucleotide sequence in FIG. 8.The encoded protein, human B7-2, is predicted to be 329 amino acids inlength (SEQ ID NO:2). This protein sequence exhibits many featurescommon to other type 1 Ig superfamily embrane proteins. Proteintranslation is predicted to begin at the ATG codon (nucleotide 107-109)based on DNA homology in this region with the consensus eukaryotictranslation initiation site (Kozak, M. (1987) Nucl. Acids Res.15:8125-8148). The amino terminus of the human B7-2 protein (amino acids1 to 23) has the characteristics of a secretory signal peptide with apredicted cleavage between the alanines at positions 23 and 24 (vonHeijne (1986) Nucl. Acids Res. 14:4683). Processing at this site wouldresult in a human B7-2 membrane bound protein of 306 amino acid with anunmodified molecular weight of approximately 34 kDa. This protein wouldconsist of an extracellular Ig superfamily V and C like domains, of fromabout amino acid residue 24-245, a hydrophobic transmembrane domain offrom about amino acid residue 246-268 and a long cytoplasmic domain offrom about amino acid residue 269-329. The homologies to the Igsuperfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 218. The extracellular domain also contains eight potentialN-linked glycosylation sites. E coli transfected with a vectorcontaining the cDNA insert of clone 29, encoding the human B7-2 protein,was deposited with the American Type Culture Collection (ATCC) on Jul.26, 1993 as Accession No. 69357.

[0224] Comparison of both the nucleotide and amino acid sequences ofhuman B7-2 with the GenBank and EMBL databases showed that only thehuman and murine B7-1 proteins are related. Alignment of the three B7protein sequences (see FIG. 13) shows that human B7-2 has approximately26% amino acid identity with human B7-1. FIG. 13 represents thecomparison of the amino acid sequences for human B7-2 (hB7-2) (SEQ IDNO:2), human B7-1 (hB7-1) (SEQ ID NO: 28 and 29) and murine B7 (mB7)(SEQ ID NO: 30 and 31). The amino acid sequences for the human B7-1 andmurine B7 (referred to herein as murine B7-1) can be found in Genbank atAccession #M27533 and X60958 respectively. Vertical lines in FIG. 13show identical amino acids between the hB7-2 and hB7-1 or mB7. Identicalamino acids between hB7-1 and mB7 are not shown. The hB7-2 proteinexhibits the same general structure as hB7-1 as defined by the commoncysteines (positions 40 and 110, IgV domains; positions 157 and 217, IgCdomain) which the Ig superfamily domains and by many other common aminoacids. Since both hB7-1 and mB7 have been shown to bind to both humanCTLA4 and human CD28, the amino acids in common between these tworelated proteins will be those necessary to comprise a CTLA4 or CD28binding sequence. An example of such a sequence would be the KYMGRTSFD(position 81-89, hB7-2) (SEQ ID NO:17) or KSQDNVTELYDVS (position188-200, hB7-2) (SEQ ID NO:18). Additional related sequences are evidentfrom the sequence comparison and others can be inferred by consideringhomologous related amino acids such as aspartic acid and glutamic acid,alanine and glycine and other recognized functionally related aminoacids. The B7 sequences share a highly positive charged domain with thecytoplasmic portion WKWKKKKRPRNSYKC (position 269-282, hB7-2) (SEQ IDNO:19) which is probably involved in intracellular signaling.

EXAMPLE 5 Characterization of the Recombinant B7-2 Antigen

[0225] A. B7-2 Binds CTLA4Ig and not Anti-B7-1 and Anti-B7-3 MonoclonalAntibodies

[0226] COS cells transfected with either vector DNA (pCDNAI), or anexpression plasmid containing B7-1 (B7-1) or B7-2 (B7-2) were prepared.After 72 hours, the transfected COS cells were detached by incubation inPBS containing 0.5 mM EDTA and 0.02% Na azide for 30 min. at 37° C.Cells were analyzed for cell surface expression by indirectimmunofluorescence and flow cytometric analysis using fluorosceinisothiocyanate conjugated (FITC) goat-anti-mouse Ig or goat-anti-humanIgG FITC (FIG. 9). Cell surface expression of B7-1 was detected withmAbs 133 (anti-B7-1) and BB-1 (anti-B7-1 and anti-B7-3) and withCTLA4Ig, whereas B7-2 reacted only with CTLA4Ig. Neither of the B7transfectants showed any staining with the isotype controls (IgM orcontrol Ig). The vector transfected COS cells showed no staining withany of the detection reagents. In addition, none of the cells showed anystaining with the FITC labeled detection reagents and alone. Thisdemonstrates that B7-2 encodes a protein that is a CTLA4counter-receptor but is distinct from B7-1 and B7-3,

[0227] B. RNA Blot Analysis of B7-2 Expression in Unstimulated andActivated Human B Cells. Cell Lines, and Myelomas

[0228] Human splenic B cells were isolated by removing T cells andmonocytes as previously described (Freedman, A. S., Freeman, G. J.,Horowitz, J. C., Daley, J., Nadler, L. M., J. Immunol. (1987)137:3260-3267). Splenic B cells were activated using anti-Ig beads andcells were harvested at the indicated times (Freedman et al., (1987),cited supra). Human myelomas from bone marrow specimens were enriched byremoving T cells and monocytes using E rosettes and adherence aspreviously described (Freeman, G. J., et al., J. Immunol. (1989)143:2714-2722). RNA was prepared by guanidine thiocyanate homogenizationand cesium chloride centrifugation. Equal amounts of RNA (20 μg) wereelectrophoresed on an agarose gel, blotted, and hybridized to³²P-labelled B7-2 cDNA. FIG. 10, panel a, shows RNA blot analysis ofunstimulated and anti-Ig activated human splenic B cells and of celllines including Raji (B cell Burkitts lymphoma), Daudi (B cell Burkitt'slymphoma), RPMI 8226 (myeloma), K562 (erythroleukemia), and REX (T cellacute lymphoblastic leukemia). FIG. 10, panel b shows RNA blot analysisof human myeloma specimens.

[0229] Three mRNA transcripts of 1.35, 1.65 and 3.0 kb were identifiedby hybridization to the B7-2 cDNA (FIG. 10, panel b). RNA blot analysisdemonstrated that B7-2 mRNA is expressed in unstimulated human splenic Bcells and increases 4-fold following activation (FIG. 10, panel a). B7-2mRNA was expressed in B cell neoplastic lines (Raji, Daudi) and amyeloma (RPMI 8226) but not in the erythroleukemia K562 and the T cellline REX. In contrast, we have previously shown that B7-1 mRNA is notexpressed in resting B cells and is transiently expressed followingactivation (G. J. Freeman et al. (1989) supra). Examination of mRNAisolated from human myelomas demonstrates that B7-2 mRNA is expressed in6 of 6 patients, whereas B7-1 was found in only 1 of these 6 (G. J.Freeman et al. (1989) supra). Thus, B7-1 and B7-2 expression appears tobe independently regulated.

[0230] C. Costimulation

[0231] Human CD28+ T cells were isolated by immunomagnetic beaddepletion using monoclonal antibodies directed against B cells, naturalkiller cells and macrophages as previously described (Gimmi, C. D., etal. (1993) Proc. Natl. Acad. Sci. USA 9, 6586-6590). B7-1, B7-2 andvector transfected COS cells were harvested 72 hours after transfection,incubated with 25 μg/ml of mitomycin-C for 1 hour, and then extensivelywashed. 10⁵ CD28⁺ and T cells were incubated with 1 ng/ml of phorbolmyristic acid (PMA) and the indicated number of COS transfectants (FIG.11). As shown in FIG. 11, panel a, T cell proliferation was measured by3H-thymidine (1 μCi) incorporated for the last 12 hours of a 72 hourincubation. Panel b of FIG. 11 shows IL-2 production by T cells asmeasured by ELISA (Biosource, CA) using supernatants harvested 24 hoursafter the initiation of culture.

[0232] D. B7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-B7-3mAbs but is Blocked by CTLA4-Ig and Anti-CD28 Fab

[0233] Human CD28⁺ T cells were isolated by immunomagnetic beaddepletion using mAbs directed against B cells, natural killer cells, andmacrophages as previously described (Gimmi, C. D., Freeman, G. J.,Gribben, J. G., Gray, G., Nadler, L. M. (1993) Proc. Natl. Acad. Sci USA9, 6586-6590). B7-1, B7-2, and vector transfected COS cells wereharvested 72 hours after transfection, incubated with 25 μg/ml ofmitomycin-C for 1 hour, and then extensively washed. 10⁵ CD28⁺ T cellswere incubated with 1 ng/ml of phorbol myristic acetate (PMA) and 2×10⁴COS transfectants. Blocking agents (10 μg/nl) are indicated on the leftside of FIG. 12 and include: 1) no monoclonal antibody (no blockingagents), 2) mAb 133 (anti-B7-1 mAb), 3) mAb BB1 (anti-B7-1 and anti-B7-3mAb), 4) mAb B5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6)CTLA-Ig, and 7) control Ig. Panel a of FIG. 12 shows proliferationmeasured by ³H-thymidine (1 μCi) incorporation for the last 12 hours ofa 72 hour incubation. FIG. 12, panel b, shows IL-2 production asmeasured by ELISA (Biosource, CA) using supernatants harvested 24 hoursafter the initiation of culture.

[0234] B7-1 and B7-2 transfected COS cells costimulated equivalentlevels of T cell proliferation when tested at various stimulator toresponder ratios (FIG. 11). Like B7-1, B7-2 transfected COS cellcostimulation resulted in the production of IL-2 over a wide range ofstimulator to responder cell ratios (FIG. 11). In contrast, vectortransfected COS cells did not costimulate T cell proliferation or IL-2production.

[0235] E. B7-2 Costimulation is not Blocked by Anti-B7-1 and Anti-B7-3mAbs but is Blocked by CTLA4-Ig and Anti-CD28 Fab

[0236] Human CD28⁺ T cells were isolated by immunomagnetic beaddepletion using mAbs directed against B cells, natural killer cells, andmacrophages as previously described (Gimmi, C. D., Freeman, G. J.,Gribben, J. G., Gray, G., Nadler, L. M. (1993) Proc. Natl. Acad. Sci USA9, 6586-6590). B7-1, B7-2, and vector transfected COS cells wereharvested 72 hours after transfection, incubated with 25 μg/ml ofmitomycin-C for 1 hour, and then extensively washed. 10⁵ CD28⁺ T cellswere incubated with 1 ng/ml of phorbol myristic acetate (PMA) and 2×10⁴COS transfectants. Blocking agents (10 μg/ml) are indicated on the leftside of FIG. 12 and include: 1) no monoclonal antibody (no blockingagents), 2) mAb 133 (anti-B7-1 mAb), 3) mAb BB1 (anti-B7-1 and anti-B7-3mAb), 4) mAb B5 (control IgM mAb), 5) anti-CD28 Fab (mAb 9.3), 6)CTLA-Ig, and 7) control Ig. Panel a of FIG. 12 shows proliferationmeasured by ³H-thymidine (1 μCi) incorporation for the last 12 hours ofa 72 hour incubation. FIG. 12, panel b, shows IL-2 production asmeasured by ELISA (Biosource, CA) using supernatants harvested 24 hoursafter the initiation of culture.

[0237] To distinguish B7-2 from B7-1 and B7-3, mAbs directed againstB7-1 and B7-3 were used to inhibit proliferation and IL-2 production ofsubmitogenically activated human CD28⁺ T cells. Both B7-1 and B7-2 COStransfectants costimulated T cell proliferation and IL-2 production(FIG. 12). MAbs 133 (Freedman, A. S. et al. (1987) supra) (anti-B7-1)and BB1 (Boussiotis, V. A., et al., (in review) Proc. Natl. Acad. Sci.USA; Yokochi, T., Holly, R. D., Clark, E. A. (1982) J. Immunol. 128,823-827) (anti-B7-1 and anti-B7-3) completely inhibited proliferationand IL-2 secretion induced by B7-1 but had no effect upon costimulationby B7-2 transfected COS cells. Isotype matched control B5 mAb had noeffect. To determine whether B7-2 signals via the CD28/CTLA4 pathway,anti-CD28 Fab and CTLA4-Ig fusion protein were tested to determinewhether they inhibited B7-2 costimulation. Both anti-CD28 Fab andCTLA4-Ig inhibited proliferation and IL-2 production induced by eitherB7-1 or B7-2 COS transfectants whereas control Ig fusion protein had noeffect (FIG. 12). While CTLA4-Ig inhibited B7-2 costimulation ofproliferation by only 90%, in other experiments inhibition was morepronounced (98-100%). None of the blocking agents inhibited T cellproliferation or IL-2 production induced by the combination of PMA andphytohemagglutinin.

[0238] Like B7-1, B7-2 is a counter-receptor for the CD28 and CTLA4 Tcell surface molecules. Both proteins are similar in that they are: 1)expressed on the surface of APCs; 2) structurally related to the Igsupergene family with an IgV and IgC domain which share 26% amino acididentity, and 3) capable of costimulating T cells to produce IL-2 andproliferate. However, B7-1 and B7-2 differ in several fundamental ways.First, B7-2 mRNA is constitutively expressed in unstimulated B cells,whereas B7-1 mRNA does not appear until 4 hours and cell surface proteinis not detected until 24 hours (Freedman, A. S., et al. (1987) supra;Freeman, G. J., et al. (1989) supra). Unstimulated human B cells do notexpress CTLA4 counter-receptors on the cell surface and do notcostimulate T cell proliferation (Boussiotis, V. A., et al. supra).Therefore, expression of B7-2 mRNA in unstimulated B cells would allowrapid expression of B7-2 protein on the cell surface followingactivation, presumably from stored mRNA or protein. Costimulation byB7-2 transfectants is partially sensitive to paraformaldehyde fixation,whereas B7-2 costimulation is resistant (Gimmi, C. D., et al. (1991)Proc. Natl. Acad. Sci. USA 88, 6575-6579). Second, expression of B7-1and B7-2 in cell lines and human B cell neoplasms substantially differs.Third, B7-2 protein contains a longer cytoplasmic domain than B7-1 andthis could play a role in signaling B-cell differentiation. Thesephenotypic and function al differences suggest that these homologousmolecules may have biologically distinct function s.

EXAMPLE 6 Cloning and Sequencing of the Murine B7-2 Antigen

[0239] A. Construction of cDNA Library

[0240] A cDNA library was constructed in the pCDM8 vector (Seed, Nature,329:840 (1987)) using poly(A)⁺ RNA from dibutryl cyclic AMP (cAMP)activated M12 cells (a murine B cell tumor line) as described (Aruffo etal, Proc. Natl. Acad. Sci USA, 84:3365 (1987)).

[0241] M12 cells were cultured at 1×10⁶ cells/ml in complete culturemedia, {RPMI 1640 with 10% heat inactivated fetal calf serum (FCS), 2 mMglutamine, 1 mM sodium pyruvate, penicillin (100 units/ml), streptomycinsulfate (100 μg/ml) and gentamycin sulfate (5 μg/ml)}, in tissue cultureflasks and were activated by 300 μg/ml dibutryl cAMP (Nabavi, N., et al.(1992) Nature 360, 266-268). Activated M12 cells were harvested after 0,6, 12, 18, 24 and 30 hours.

[0242] RNA was prepared by homogenizing activated M12 cells in asolution of 4M guanidine thiocyanate, 0.5% sarkosyl, 25 mM EDTA, pH 7.5,0.13% Sigma anti-foam A, and 0.7% mercaptoethanol. RNA was purified fromthe homogenate by centrifugation for 24 hour at 32,000 rpm through asolution of 5.7M CsCl, 10 mM EDTA, 25 mM Na acetate, pH 7. The pellet ofRNA was dissolved in 5% sarkosyl, 1 mM EDTA, 10 mM Tris, pH 7.5 andextracted with two volumes of 50% phenol, 49% chloroform, 1% isoamylalcohol. RNA was ethanol precipitated twice. Poly(A)⁺ RNA used in cDNAlibrary construction was purified by two cycles of oligo (dT)-celluloseselection

[0243] Complementary DNA was synthesized from 5.5 μg of dibutryl cAMPactivated murine M12 cell poly(A)⁺ RNA in a reaction containing 50 mMTris, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 500 μM dATP,dCTP, dGTP, dTTP, 50 μg/ml oligo(dT)₁₂₋₁₈, 180 units/ml RNasin, and10,000 units/ml Moloney-MLV reverse transcriptase in a total volume of55 μl at 37° C. for 1 hr. Following reverse transcription, the cDNA wasconverted to double-stranded DNA by adjusting the solution to 25 mMTris, pH 8.3, 100 mM KCl, 5 mM MgCl₂, 250 μM each dATP, dCTP, dGTP,dTTP, 5 mM dithiothreitol, 250 units/ml DNA polymerase I, 8.5 units/mlribonuclease H and incubating at 16° C. for 2 hr. EDTA was added to 18mM and the solution was extracted with an equal volume of 50% phenol,49% chloroform, 1% isoamyl alcohol. DNA was precipitated with twovolumes of ethanol in the presence of 2.5M ammonium acetate and with 4micrograms of linear polyacrylamide as carrier. Following reversetranscription, the reverse transcriptase was inactivated by heating at70° C. for 10 min. The cDNA was converted to double-stranded DNA byadding 320 μl H₂O and 80 μl of a solution of 0.1M Tris, pH 7.5, 25 mMMgCl₂, 0.5M KCl, 250 μg/ml bovine serum albumin, and 50 mMdithiothreitol, and adjusting the solution to 200 μM each dATP, dCTP,dGTP, dTTP, 50 units/ml DNA polymerase I, 8 units/ml ribonuclease H andincubating at 16° C. for 2 hours. EDTA was added to 18 mM and thesolution was extracted with an equal volume of 50% phenol, 49%chloroform, 1% isoamyl alcohol. DNA was precipitated with two volumes ofethanol in the presence of 2.5M ammonium acetate and with 4 microgramsof linear polyacrylamide as carrier.

[0244] 2 μg of non-selfcomplementary BstXI adaptors were added to theDNA as follows: The double-stranded cDNA from 5.5 μg of poly(A)⁺ RNA wasincubated with 3.6 μg of a kinased oligonucleotide of the sequenceCTTTAGAGCACA (SEQ ID NO:15) and 2.4 μg of a kinased oligonucleotide ofthe sequence CTCTAAAG (SEQ ID NO:16) in a solution containing 6 mM Tris,pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovine serum albumin, 7 mMmercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1 mM spermidine, and600 units T4 DNA ligase in a total volume of 0.45 ml at 15° for 16hours. EDTA was added to 34 mM and the solution was extracted with anequal volume of 50% phenol, 49% chloroform, 1% isoamyl alcohol. DNA wasprecipitated with two volumes of ethanol in the presence of 2.5Mammonium acetate.

[0245] DNA larger than 600 bp was selected as follows: The adaptored DNAwas redissolved in 10 mM Tris, pH 8, 1 mM EDTA, 600 mM NaCl, 0.1%sarkosyl and chromatographed on a Sepharose CL-4B column in the samebuffer. DNA in the void volume of the column (containing DNA greaterthan 600 bp) was pooled and ethanol precipitated.

[0246] The pCDM8 vector was prepared for cDNA cloning by digestion withBstXI and purification on an agarose gel. Adaptored DNA from 5.5 μg ofpoly(A)⁺RNA was ligated to 2.25 μg of BstXI cut pCDM8 in a solutioncontaining 6 mM Tris, pH 7.5, 6 mM MgCl₂, 5 mM NaCl, 350 μg/ml bovineserum albumin, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM dithiothreitol, 1mM spermidine, and 600 units T4 DNA ligase in a total volume of 1.5 mlat 15° for 24 hr. The ligation reaction mixture was transformed intocompetent E.coli MC1061/P3 and a total of 200×10⁶ independent cDNAclones were obtained.

[0247] Plasmid DNA was prepared from a 500 ml culture of the originaltransformation of the cDNA library. Plasmid DNA was purified by thealkaline lysis procedure followed by twice banding in CsCl equilibriumgradients (Maniatis et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, N.Y. (1987)).

[0248] B. Cloning Procedure

[0249] In the first round of screening, thirty 100 mm dishes of 50%confluent COS cells were transfected with 0.05 μg/ml activated M12murine B cell library DNA using the DEAE-Dextran method (Seed et al,Proc. Natl. Acad. Sci. USA, 84:3365 (1987)). The cells were trypsinizedand re-plated after 24 hours. After 47 hours, the cells were detached byincubation in PBS/0.5 mM EDTA, pH 7.4/0.02% Na azide at 37° C. for 30min. The detached cells were treated with 10 μg/ml/human CTLA4Ig andmurine CD28Ig for 45 minutes at 4° C. Cells were washed and distributedinto panning dishes coated with affinity-purified Goat anti-human IgGantibody and allowed to attach at room temperature. After 3 hours, theplates were gently washed twice with PBS/0.5 mM EDTA, pH 7.4/0.02% Naazide, 5% FCS and once with 0.15M NaCl, 0.01 M Hepes, pH 7.4, 5% FCS.Episomal DNA was recovered from the panned cells and transformed into E.coli DH10B/P3. The plasmid DNA was re-introduced into COS cells viaspheroplast fusion as described (Seed et al, Proc. Natl. Acad. Sci. USA,84:3365 (1987)) and the cycle of expression and panning was repeatedtwice. In the second and third rounds of selection, after 47 hours, thedetached COS cells were first incubated with α-murine B7-1 mAb (16-10A1,10 μg/ml), and COS cells expressing B7-1 were removed by α-mouse IgG andIgM coated magnetic beads. COS cells were then treated with 10 μg/ml ofhuman CTLA4Ig and murine CD28Ig and murine B7-2 expressing COS cellswere selected by panning on dishes coated with goat anti-human IgGantibody. After the third round, plasmid DNA was prepared fromindividual colonies and transfected into COS cells by the DEAE-Dextranmethod. Expression of B7-2 on transfected COS cells was analyzed byindirect immunofluorescence with CTLA4Ig.

[0250] After the final round of selection, plasmid DNA was prepared fromindividual colonies. A total of 6 of 8 candidate clones contained a cDNAinsert of approximately 1.2 kb. Plasmid DNA from these eight clones wastransfected into COS cells. All six clones with the 1.2 Kb cDNA insertwere strongly positive for B7-2 expression by indirectimmunofluorescence using CTLA4Ig and flow cytometric analysis.

[0251] C. Sequencing

[0252] The B7-2 cDNA insert in clone4 was sequenced in the pCDM8expression vector employing the following strategy. Initial sequencingwas performed using sequencing primers T7, CDM8R (Invitrogen) homologousto pCDM8 vector sequences adjacent to the cloned B7-2 cDNA (see TableII). Sequencing was performed using dye terminator chemistry and an ABIautomated DNA sequencer. (ABI, Foster City, Calif.). DNA sequenceobtained using these primers was used to design additional sequencingprimers (see Table II). This cycle of sequencing and selection ofadditional primers was continued until the murine B7-2 cDNA wascompletely sequenced on both strands. TABLE II T7(F) (SEQ ID NO:3)5′d[TAATACGACTCACTATAGGG]3′ CDM8(R) (SEQ ID NO:4)5′d[TAAGGTTCCTTCACAAAG]3′ MBX4-1F (SEQ ID NO:24)5′d[ACATAAGCCTGAGTGAGCTGG]3′ MBX4-2R (SEQ ID NO:25)5′d[ATGATGAGCAGCATCACAAGG]3′ MBX4-14 (SEQ ID NO:26)5′d[TGGTCGAGTGAGTCCGAATAC]3′ MBX4-2F (SEQ ID NO:27)5′d[GACGAGTAGTAACATACAGTG]3′

[0253] A murine B7-2 clone (mB7-2, clone 4) was obtained containing aninsert of 1,163 base pairs with a single long open reading frame of 927nucleotides and approximately 126 nucleotides of 3′ noncoding sequences(FIG. 14, SEQ ID NO:22). The predicted amino acid sequence encoded bythe open reading frame of the protein is shown below the nucleotidesequence in FIG. 14. The encoded murine B7-2 protein, is predicted to be309 amino acid residues in length (SEQ ID NO:23). This protein sequenceexhibits many features common to other type I Ig superfamily membraneproteins. Protein translation is predicted to begin at the methioninecodon (ATG, nucleotides 111 to 113) based on the DNA homology in thisregion with the consensus eucaryotic translation initiation site (seeKozak, M. (1987) Nucl. Acids Res. 11:8125-8148). The amino terminus ofthe murine B7-2 protein (amino acids 1 to 23) has the characteristics ofa secretory signal peptide with a predicted cleavage between the alanineat position 23 and the valine at position 24 (von Heijne (1987) Nucl.Acids Res. 14:4683). Processing at this site would result in a murineB7-2 membrane bound protein of 286 amino acids having an unmodifiedmolecular weight of approximately 32 kDa. This protein would consist ofan approximate extracellular Ig superfamily V and C like domains of fromabout amino acid residue 24 to 246, a hydrophobic transmembrane domainof from about amino acid residue 247 to 265, and a long cytoplasmicdomain of from about amino acid residue 266 to 309. The homologies tothe Ig superfamily are due to the two contiguous Ig-like domains in theextracellular region bound by the cysteines at positions 40 to 110 and157 to 216. The extracellular domain also contains nine potentialN-linked glycosylation sites and, like murine B7-1, is probablyglycosylated. Glycosylation of the murine B7-2 protein may increase themolecular weight to about 50-70 kDa. The cytoplasmic domain of murineB7-2 contains a common region which has a cysteine followed bypositively charged amino acids which presumably functions as signalingor regulatory domain within an APC. Comparison of both the nucleotideand amino acid sequences of murine B7-2 with the GenBank and EMBLdatabases yielded significant homology (about 26% amino acid sequenceidentity) with human and murine B7-1. Murine B7-2 exhibits about 50%identity and 67% similarity with its human homologue, hB7-2. E. coli(DH106/p3) transfected with a vector (plasmid pmBx4) containing a cDNAinsert encoding murine B7-2 (clone 4) was deposited with the AmericanType Culture Collection (ATCC) on Aug. 18, 1993 as Accession No. 69388.

[0254] D. Costimulation

[0255] CD4⁺ murine T cells were purified by first depleting red bloodcells by treatment with Tris-NH₄Cl. T cells were enriched by passageover a nylon wool column. CD4⁺ T cells were purified by two-foldtreatment with a mixture of anti-MHC class II and anti-CD28 mAbs andrabbit complement. Murine B7-1 (obtained from Dr. Gordon Freeman,Dana-Farber Cancer Institute, Boston, Mass.; see also, Freeman, G. J. etal (1991) J. Exp. Med. 174, 625-631) murine B7-2, and vector transfectedCOS cells were harvested 72 hours after tmasfection, incubated with 25μg/ml mitomycin-C for one hour, and then extensively washed. 10⁵ murineCD4⁺ T cells were incubated with 1 ng/ml of phorbol myristic acid (PMA)and 2×10⁴ COS transfectants (Table III). T cell proliferation wasmeasured by ³H-thymidine (1 μCi) incorporated for the last 12 hours of a72 hour incubation. TABLE III 3H-Thymidine Incorporation (cpm) CD4⁺ Tcells  175 CD4⁺ T cells + 1 ng/ml PMA   49 CD4⁺ T cells + COS-vector 1750 CD4⁺ T cells + COS-B7-1  4400 CD4⁺ T cells + COS-B7-2  2236 CD4⁺ Tcells + 1 ng/ml PMA + COS-vector  2354 CD4⁺ T cells + 1 ng/ml PMA +COS-B7-1 67935 CD4⁺ T cells + 1 ng/ml PMA + COS-B7-2 43847

EXAMPLE 7 Construction and Characterization of Human B7-2 ImmunoglobulinFusion Proteins

[0256] A. Preparation of Human B7-2Ig Fusion Proteins

[0257] The extracellular portion of human B7-2 was prepared as a fusionprotein coupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications including those whichreduce or eliminate effector activity inherent in the immunoglobulinstructure. Briefly, DNA encoding the extracellular portion of hB7-2 wasjoined to DNA encoding the hinge, CH2 and CH3 regions of human IgCγ1 orIgCγ4 modified by directed mutagenesis. This was accomplished asdescribed in the following subsections.

[0258] B. Preparation of Gene Fusions

[0259] DNA fragments corresponding to the DNA sequences of interest wereprepared by polymerase chain reaction (PCR) using primer pairs describedbelow. In general, PCR reactions were prepared in 100 μl final volumecomposed of Taq, polymerase buffer (Gene Amp PCR Kit,Perkin-Elmer/Cetus, Norwalk, Conn.) containing primers (1 μM each),dNTPs (200 μM each) 1 ng of template DNA, and Taq, polymerase (Saiki, R.K., et al. (1988) Science 239:487-491). PCR DNA amplifications were runon a thermocycler (Ericomp, San Diego, Calif.) for 25 to 30 cycles eachcomposed of a denaturation step (1 minute at 94° C.), a renaturationstep (30 seconds at 54° C.), and a chain elongation step (1 minute at72° C.). The structure of each hB7-2 Ig genetic fusion consisted of asignal sequence to facilitate secretion coupled to the extracellulardomain of B7-2 and the hinge, CH2 and CH3 domains of human IgCγ1 orIgCγ4. The IgC gamma 1 and IgC gamma 4 sequences contained nucleotidechanges within the hinge region to replace cysteine residues availablefor disulfide bond formation with serine residues and may containnucleotide changes to replace amino acids within the CH2 domain thoughtto be required for IgC binding to Fc receptors and complementactivation.

[0260] Sequence analysis confirmed structures of both mγ₄ and γ₁ clones,and each construct was used to transfect 293 cells to test transientexpression. hIgG ELISA measured/confirmed transient expression levelsapproximately equal to 100 ng protein/ml cell supernatant for bothconstructs. NSO cell lines were transfected for permanent expression thethe fuision proteins.

[0261] C. Genetic Construction of hB7-2Ig Fusion Proteins

[0262] (1). Preparation of Signal Sequence

[0263] PCR amplification was used to generate an immunoglobulin signalsequence suitable for secretion of the B7-2Ig fusion protein frommammalian cells. The Ig signal sequence was prepared from a plasmidcontaining the murine IgG heavy chain gene (Orlandi, R. et al. (1989)Proc. Natl. Acad. Sci. USA. 86:38333837) using the oligonucleotide5′-GGCACTAGGTCTCCAGCTTGAGATCACAGTTCTCTCTAC-3′ (#01) (SEQ ID NO: ) as theforward primer and the oligonucleotide5′-GCTTGAATCTTCAGAGGAGCGGAGTGGACACCTGTGG-3′ (#02) (SEQ ID NO: ) as thereverse PCR primer. The forward PCR primer (SEQ ID NO: ) containsrecognition sequences for restriction enzymes BsaI and is homologous tosequences 5′ to the initiating methionine of the Ig signal sequence. Thereverse PCR primer (SEQ ID NO: ) is composed of sequences derived fromthe 5′ end of the extracellular domain of hB7-2 and the 3′ end of the Igsignal sequence. PCR amplification of the murine Ig signal template DNAusing these primers resulted in a 224 bp product which is composed ofBsaI restriction sites followed by the sequence of the Ig signal regionfused to the first 20 nucleotides of the coding sequence of theextracellular domain of hB7-2. The junction between the signal sequenceand hB7-2 is such that protein translation beginning at the signalsequence will continue into and through hB7-2 in the correct readingframe.

[0264] (2). Preparation of the hB7-2 Gene Segment

[0265] The extracellular domain of the hB7.2 gene was prepared by PCRamplification of plasmid containing the hB7-2 cDNA inserted intoexpression vector pCDNAI (Freeman et al., Science 262:909-11 (1994)):

[0266] The extracellular domain of hB7-2 was prepared by PCRamplification using oligonucleotide 5′-GCTCCTCTGAAGATTCAAGC-3′ (#03)(SEQ ID NO: ) as the forward primer and oligonucleotide5′-GGCACTATGATCAGGGGGAGGCTGAGGTCC-3′ (#04) (SEQ ID NO: ) as the reverseprimer. The forward PCR primer contained sequences corresponding to thefirst 20 nucleotides of the B7-2 extracellular domain and the reversePCR primer contained sequences corresponding to the last 22 nucleotidesof the B7-2 extracellular domain followed by a Bcl I restriction siteand 7 noncoding nucleotides. PCR amplification with primer #03 and #04yields a 673 bp product corresponding to the extracellular IgV and IgClike domains of hB7-2 followed by a unique Bcl I restriction site.

[0267] The signal sequence was attached to the extracellular portion ofhB7-2 by PCR as follows. DNA-PCR products obtained above correspondingto the signal sequence and the hB7-2 extracellular domain were mixed inequimolar amounts, denatured by heating to 100° C., held at 54° C. for30° C. to allow the complementary ends to anneal and the strands werefilled in using dNTPs and Toq polymerase. PCR primers #01 and #04 wereadded and the entire fragment produced by PCR amplification to yield a˜880 fragment composed of a BsaI restriction site followed by the signalsequence fused to the extracellular domain of hB7-2, followed by a Bcl Irestriction site.

[0268] (3). Cloning and Modification of Immunoglobulin Fusion Domain

[0269] Plasmid pSP721gGl was prepared by cloning the 2000 bp segment ofhuman IgGl heavy chain genomic DNA (Ellison, J. W., et al. (1982) Nucl.Acids. Res. 10:4071-4079) into the multiple cloning site of cloningvector pSP72 (Promega, Madison, Wis.). Plasmid pSP721gGl containedgenomic DNA encoding the CH1, hinge, CH2 and CH3 domains of the heavychain human Igγ1 gene. PCR primers designed to amplify the hinge-CH2-CH3portion of the heavy chain along with the intervening DNA were preparedas follows. The forward PCR primer5′-GCATTTTAAGCTTTTTCCTGATCAGGAGCCCAAATCTTCTGACAAAACTCACACATCTCCACCGTCTCCAGGTAAGCC-3′ (SEQ ID NO: ) containedHindIII and Bcl I restriction sites and was homologous to the hingedomain sequence except for five nucleotide substitutions which wouldchange the three cysteine residues to serines. The reverse PCR primer5′TAATACGACTCACTATAGGG-3′ (SEQ ID NO: ) was identical to thecommercially available T7 primer (Promega, Madison, Wis.). Amplificationwith these primers yielded a 1050 bp fragment bounded on the 5′ end byHindIII and BclI restriction sites and on the 3′ end by BamHl, Smal,Kpnl, Sacl, EcoR1, Clal, EcoR5 and Bglll restriction sites. Thisfragment contained the IgC hinge domain in which the three cysteinecodons had been replaced by serine codons followed by an intron, the CH2domain, an intron, the CH3 domain and additional 3′ sequences. After PCRamplification, the DNA fragment was digested with Hindlll and EcoR1 andcloned into expression vector pNRDSH digested with the same restrictionenzymes. This created plasmid pNRDSH/IgG1.

[0270] A similar PCR based strategy was used to clone the hinge-CH2-CH3domains of human IgCgamma4 constant regions. A plasmid, p428D (MedicalResearch Council, London, England) containing the complete IgCgamma4heavy chain genomic sequence (Ellison, J. Buxbaum, J. and Hood, L. E.(1981) DNA 1: 11-18) was used as a template for PCR amplification usingoligonucleotide 5′GAGCATTTTCCTGATCAGGAGTCCAAATATGGTCCCCCATCCCATCATCCCCAGGTAAGCCAACCC-3′ (SEQ ID NO: ) as theforward PCR primer and oligonucleotide5′GCAGAGGAATCGAGCTCGGTACCCGGGGATCCCCAGTGTGGGGACAGTGGGA CCGCTCTGCCTCCC-3′(SEQ ID NO: ) as the reverse PCR primer. The forward PCR primer (SEQ IDNO: ) contains a Bcll restriction site followed by the coding sequencefor the hinge domain of IgCgamma4. Nucleotide substitutions have beenmade in the hinge region to replace the cysteines residues with serines.The reverse PCR primer (SEQ ID NO. ) contains a PspAI restriction site(5′CCCGGG-3′). PCR amplification with these primers results in a 1179 bpDNA fragment. The PCR product was digested with Bcll and PspAI andligated to pNRDSH/IgG1 digested with the same restriction enzymes toyield plasmid pNRDSH/IgG4. In this reaction, the IgCγ 4 domain replacedthe Igγ1 domain present in pNRDSH/IgG1.

[0271] Modification of the CH2 domain in IgC to replace amino acidsthought to be involved in binding to Fc receptor was accomplished asfollows. Plasmid pNRDSH/IgG1 served as template for modifications of theIgγ1 CH2 domain and plasmid pNRDSH/IgG4 served as template formodifications of the IgCγ 4 CH2 domain. Plasmid pNRDSH/IgG1 was PCRamplified using a forward PCR primer (SEQ ID NO: ) and oligonucleotide5′-GGGTTTT GGGGGGAAGAGGAAGACTGACGGTGCCCCC TCGGCTTCAGGTGCTGAGGAAG-3′ (SEQID NO: ) as the reverse PCR primer. The forward PCR primer (SEQ ID NO: )has been previously described and the reverse PCR primer (SEQ ID NO: )was homologous to the amino terminal portion of the CH2 domain of IgG1except for five nucleotide substitutions designed to change amino acids234, 235, and 237 (Canfield, S. M. and Morrison, S. L. (1991) J. Exp.Med. 173: 1483-1491.) from Leu to Ala, Leu to Glu, and Gly to Ala,respectively. Amplification with these PCR primers will yield a 239 bpDNA fragment consisting of a modified hinge domain, an intron andmodified portion of the CH2 domain. Plasmid pNRDSH/IgG1 was also PCRamplified with the oligonucleotide5′-CATCTCTTCCTCAGCACCTGAAGCCGAGGGGGCACCGTCAGTCTTCCTCTTCCC CC-3′ (SEQ IDNO: ) as the forward primer and oligonucleotide (SEQ ID NO: ) as thereverse PCR primer. The forward PCR primer (SEQ ID NO: ) iscomplementary to primer (SEQ ID NO: ) and contains the fivecomplementary nucleotide changes necessary for the CH2 amino acidreplacements. The reverse PCR primer (SEQ ID NO: ) has been previouslydescribed. Amplification with these primes yields a 875 bp fragmentconsisting of the modified portion of the CH2 domain, an intron, the CH3domain, and 3′ additional sequences. The complete Igγ1 segmentconsisting of modified hinge domain, modified CH2 domain and CH3 domainwas prepared by an additional PCR reaction. The purified products of thetwo PCR reactions above were mixed, denatured (95° C., 1 minute) andthen renatured (54° C., 30 seconds) to allow complementary ends of thetwo fragments to anneal. The strands were filled in using dNTP and Taqpolymerase and the entire fragment amplified using forward PCR primer(SEQ ID NO: ) and reverse PCR primer (SEQ ID NO: ). The resultingfragment of 1050 bp was purified, digested with HindIII and EcoR1 andligated to pNRDSH previously digested with the same restriction enzymesto yield plasmid pNRDSHIgG1 m.

[0272] Two amino acids at immunoglobulin positions 235 and 237 werechanged from Leu to Glu and Gly to Ala, respectively, within the IgCγ4CH2 domain to eliminate Fc receptor binding. Plasmid pNRDSH/IgG4 was PCRamplified using the forward primer (SEQ ID NO: ) and the oligonucleotide5′-CGCACGTGACCTCAGGGGTCCGGGAGATCATGAGAGTGTCCTTGGGTTTTGGGGGGAACAGGAAGACTGATGGTGCCCCCTCGAACTCAGGTGCTGAGG-3′ (SEQ ID NO: ) as thereverse primer. The forward primer has been previously described and thereverse primer was homologous to the amino terminal portion of the CH2domain, except for three nucleotide substitutions designed to replacethe amino acids described above. This primer also contained a Pmllrestriction site for subsequent cloning. Amplification with theseprimers yields a 265 bp fragment composed of the modified hinge region,and intron, and the modified 5′ portion of the CH2 domain.

[0273] Plasmid pNRDSH/lgG4 was also PCR amplified with theoligonucleotide 5′-CCTCAGCACCTGAGTTCGAGGGGGCACCATCAGTCTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCG-3′ (SEQ ID NO: ) asthe forward primer and oligonucleotide (SEQ ID NO: ) as the reverse PCRprimer. The forward PCR primer (SEQ ID NO: ) is complementary to primer(SEQ ID NO: ) and contains the three complementary nucleotide changesnecessary for the CH2 amino acid replacements. The reverse PCR primer(SEQ ID NO: ) has been previously described. Amplification with theseprimes yields a 1012 bp fragment consisting of the modified portion ofthe CH2 domain, an intron, the CH3 domain, and 3′ additional sequences.The complete IgCγ4 segment consisting of modified hinge domain, modifiedCH2 domain and CH3 domain was prepared by an additional PCR reaction.The purified products of the two PCR reactions above were mixed,denatured (95° C., 1 minute) and then renatured (54° C., 30 seconds) toallow complementary ends of the two fragments to anneal. The strandswere filled in using dNTP and Taq polymerase and the entire fragmentamplified using forward PCR primer (SEQ ID NO: ) and reverse PCR primer(SEQ ID NO: ). The resulting fragment of 1179 bp was purified, digestedwith Bcll and PspAI and ligated to pNRDSH previously digested with thesame restriction enzymes to yield plasmid pNRDSH/IgG4m.

[0274] (4). Assembly of Final hB7-2Ig Genes

[0275] The PCR fragment corresponding to the Ig signal-hB7-2 gene fusionprepared above was digested with BsaI and Bcll restriction enzymes andligated to pNRDSH/IgG1, pNRDSH/IgG1m, pNRDSH/IgG4, and pNRDSH/IgG4mpreviously digested with Hind III and BclI. The ligated plasmids weretransformed into E. coli JM109 using CaC12 competent cells andtransformants were selected on L-agar containing ampicillin (50 μg/ml;Molecular Cloning: A Laboratory Manual (1982) Eds. Maniatis, T.,Fritsch, E. E., and Sambrook, J. Cold Spring Harbor Laboratory).Plasmids isolated from the transformed E. coli were analyzed byrestriction enzyme digestion. Plasmids with the expected restrictionplasmid were sequenced to verify all portions of the signal-hB7-2-IgGgene fusion segments.

[0276] D. Expression Cloning of hB7-2V-IgG1 and hB7-2C IgG1

[0277] The variable and constant domains of human B7-2 were separatelycloned into pNRDSH/IgG1. These clonings were accomplished using PCR. Theportions of hB7-2 corresponding to the variable and constant regionswere determined from intron/exon mapping and previously published genestructure analysis. Human B7-2 Variable Domain      5′GCTCCTCTGAAGATT.....................GAACTGTCAGTGCTT3′ (SEQ IDNO: )          A  P  L  K   I                E  L   S V  L    (SEQ IDNO: ) Human B7-2 Constant Domain      5′GCTAACTTCAGTCAA.....................CCTTTCTCTATAGAG3′ (SEQ IDNO: )         A  N  F  S  Q                        P  F  S  I  E      (SEQ IDNO: )

[0278] (1). Assembly of hB7-2VIg

[0279] The hB7-2V domain Ig sequence was assembled using a PCR strategysimilar to that shown above. The signal sequence was derived from theonco M gene by PCR amplification of a plasmid containing the onco M geneusing oligonucleotide5′-GCAACCGGAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACG-3′ (#05) (SEQ ID NO:) as the forward PCR primer and5′-AGTCTCATTGAAATAAGCTTGAATCTTCAGAGGAGCCATGCTGGCCATGCTTGGA AACAGGAG-3′(#06) (SWQ ID NO: ) as the reverse primer. The forward PCR primer (#05)contains a Hind III restriction site and the amino terminal portion ofthe onco M signal sequence. The reverse PCR (#06) contains the sequencecorresponding to the 3′ portion of the onco M signal sequence fused tothe 5′ end of the hB7-2 IgV like domain.

[0280] The hB7-2 IgV like domain was obtained by PCR amplification ofthe hB7-2 cDNA using oligonucleotide5′-CTCCTGTTTCCAAGCATGGCCAGCATGGCTCCTCTGAA GATTCAGGCTTATTTCAATGAGAC-3′(#07) (SEQ ID NO: ) as the forward and oligonucleotide5′-TGTGTGTGGAATTCTCATTACTGATCAAGCACTGACAGTTCAGAATTCATC-3′ (#08) (SEQ IDNO: ) as the reverse PCR primer. PCR amplification with these primersyields the hB7-2 IgV domain with a portion of the 3′ end of the onco Msignal sequence on the 5′ end and a Bcl I restriction site on the 3′end. The signal and IgV domain were linked together in a PCR reaction inwhich equimolar amounts of the onco M signal and IgV domain DNAfragments were mixed, denatured, annealed, and the strands filled in.Subsequent PCR amplification using forward primer #05 and reverse primer#08 yielded a DNA fragment containing a Hind III restriction site,followed by the onco M signal fused to the B7-2 IgV domain followed by aBcl I restriction site. This PCR fragment was digested with Hind II andBcl I and cloned into expression vector pNRDSH/IgG1 digested with thesame restriction enzymes to yield pNRDSH/B7-2CIg.

[0281] (2). Assembly of hB7-2CIg

[0282] The expression plasmid for hB7-2IgC domain was prepared asdescribed above for the IgV domain except for using PCR primers specificfor the IgC domain. The onco M signal sequence was prepared usingoligonucleotide #05 as the forward PCR primer and oligonucleotide5′-AGAAATTGGTACTATTTCAGGTTGACTGAAGTTAGCCATGCTGGCCATGCTTGGA AACAGGAG-3′(#09) (SEQ ID NO: ) as the reverse PCR primer. The hB7-2 IgC domain wasprepared using oligonucleotide5′-CTCCTGTTTCCAAGCATGGCCAGCATGGCTAACTTCAGTC AACCTGAAATAGTACCAATTTC-3′(#11) (SEQ ID NO: ) as the reverse PCR primer.

[0283] The two PCR products were mixed and amplified with primers #05and #11 to assemble the onco M signal sequence with the hB7-2IgC domain.The PCR product was subsequently digested with Hind III and BclI andligated to pNRDSH/IgG1 digested with similar restriction enzymes toyield the final expression plasmid pNRDSH/hB7-2CIgG1.

[0284] E. Competitive Binding Assays with Human B7-2Ig Fusion Proteins

[0285] To determine the affinity of binding of different forms ofsoluble B7-1 and B7-2 proteins to CTLA4, competitive binding assays wereperformed with these proteins. The soluble B7-2VIg, B7-2CIg, B7-2Ig, andB7-1Ig fusion proteins used in these assays were expressed and purifiedas follows.

[0286] The preparation of expression vectors encoding human B7-2VIg,B7-2CIg, and B7-2Ig fusion proteins is described above. The expressionvector encoding the B7-1Ig fusion protein, containing an OncoM leadersequence linked the extracellular domain of B7-1 was prepared similarly,using the PCR primers OncoMB71F (5′CTCAAGCTTGCCACCATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCGAGCATGGCGAGCATGGGTCTTTC TCACTTC3′; SEQID NO: ) and B71/BclI (5′TGTGTGTGGAATTCTCATTACTGATCAGGAAAATGCTCTTGCTTG3′; SEQ ID NO: ). The plasmid pKShB7-1,containing the OncoM leader sequence linked to the human B7-1 cDNAsequence (the nucleotide sequence of the human B7-1 cDNA is disclosed inFreeman, G. J. et al., (1989) J. Immunol. 143:2714-2722) was used as atemplate in this PCR reaction.

[0287] The B7Ig fusion proteins were prepared by transfection of COScells or Chinese Hamster Ovary (CHO) cells and purification of theprotein from the supernatant of the cultures.

[0288] Cell culture reagents were obtained from Gibco-BRL, Gaithersburg,Md. CHO cells were maintained in alpha MEM supplemented with 10% Fetalbovine serum (FBS) and glutamine. Penicillin, streptomycin, andfungizone were typically added. COS cells were maintained in DMEM with10% FBS and supplemented as described for CHO cells. All cells were keptat 5% CO2 at 37° C. in a humidified incubator.

[0289] All fusion constructs except the hB7-1Ig construct were expressedtransiently in COS cells. Typical transient transfections were doneusing 200 μgs/ml of DEAE-dextran, 100 μM chloroquine and 5 μgs of DNAper 10 cm dish in serum-free DMEM. The cells were treated until vacuoleswere noted and the cells appeared distressed (about 3 hours). Cells wereshocked with 10%DMSO/PBS for 2 minutes, then incubated with DMEM/10%FBSovernight. The following morning the media was changed to DMEM/serumfree and left until harvest at 72 hours post transfection. The hB7-1Igconstruct was transfected into CHO cells by calcium phosphatetransfection. The line was made stable using Geneticin (G418) resistanceselection, and expression was amplified using methotrexate and alpha MEMlacking nucleosides.

[0290] For all transiently transfected constructs (i.e., all constructsexcept hB7-1Ig) media enriched for Ig fusion proteins produced by thetransiently transfected host cells was harvested 72 hourspost-transfection. The amount of Ig fusion proteins produced by the hostcells was measured by performing an anti-human IgG Elisa assay with thesupernatant of the cultures. For this assay, Maxisorp plates (Nunc,Denmark) were coated overnight with 20 μgs/ml of goat anti-human IgG(H+L) (Zymed, San Francisco, Calif.) in PBS. The plates were blocked for1 hour with PBS/1.0%BSA and then incubated for an additional hour withcell culture supernatants from the transfected cells. After 5 washeswith PBS/0.05%Tween, HRP-coupled goat anti-human IgG(H+L) (Zymed) wasadded as a 1:1000 dilution in PBS. After a 1 hour incubation the plateswere washed again, then enzymatically developed using an ABTS kit(Zymed) as described above.

[0291] Expression levels were approximately 3 μg/ml for the constructs.The B7Ig fusion proteins were purified from the supernatant oftransfected host cells by protein A purification as follows. Protein ASepharose IPA300 (Repligen, Cambridge, Mass.) was washed in OBB (1.5 Mglycine, 3 M NaCl, pH 8.9), resuspended in DMEM, and added to cellculture supernatant at 1 ml/liter of supernatant. The solution was leftto mix gently overnight at 4° C. The Protein A sepharose was thenpelleted, the majority of the supernatant was removed, and the remainingsupernatant was used to resuspend the Protein-A Sepharose prior toloading onto a Poly-prep column (BioRad, Hercules, Calif.). The columnwas washed extensively with OBB and the immunoglobulin fusion proteinwas eluted in 0.1 M sodium citrate. Half volume column washes werecollected into {fraction (1/10)}th volume 1 M Tris (pH9.0). Proteincontaining fractions were identified by a standard colorimetric reaction(BioRad), pooled, and dialyzed overnight against PBS in 6000-8000 KDdialysis tubing. The purified proteins were of the expected size andhigh purity, representing >90% of total protein stained with CommassieBlue on acrylamide gels.

[0292] The ability of various B7 family-Ig fusion proteins tocompetitively inhibit the binding of biotinylated-CTLA4Ig to immobilizedB7-2Ig was determined. Competition binding assays were done as followsand analysed according to McPherson (McPherson, G. A. (1985) J.Pharmacol. Methods 14:213-228). Soluble hCTLA4Ig was labelled with ¹²⁵Ito a specific activity of approximately 2×10⁶ cpm/pmol. hB7-2-Ig fusionprotein was coated overnight onto microtiter plates at 10 mg/ml in 10 mMTris-HCl, pH8.0, 50 ml/well. The wells were blocked with binding buffer(DMEM containing 10% heat-inactivated FBS, 0.1% BSA, and 50 mM BES, pH6.8) for 2 h at room temperature. The labeled CTLA4-Ig (4 nM) was addedto each well in the presence or absence of unlabeled competing Igfulsion proteins, including fuill-length B7-2 (hB7-2Ig), full-lengthB7-1 (hB7-1Ig), the variable region-like domain of B7-2 (hB7-2VIg) andthe constant region-like domain of B7-2 (hB7-2CIg) and allowed to bindfor 2.5 h at room temperature. The wells were washed once with ice-coldbinding buffer and then four times with ice-cold PBS. Boundradioactivity was recovered by treatment of the wells with 0.5 N NaOHfor 5 min and the solubilized material removed and counted in a gammacounter.

[0293] The results of these assays are shown in FIG. 15 in which bothhB7-2Ig (10-20 mM) and hB7-2VIg (30-40 mM) competitively inhibit thebinding of CTLA4Ig to immobilized B7-2 protein. hB7-2CIg is unable tocompete with soluble CTLA4, indicating that the B7-2 binding region isin found in the variable-region like domain.

[0294] F. Competitive Binding Assays for B7-1 and B7-2 Fusion Proteins

[0295] The ability of recombinant CTLA4Ig to bind to hB7-1 or hB7-2 wasassessed in a competitive binding ELISA assay as follows. Purifiedrecombinant CTLA4Ig (20 μg/ml in PBS) was bound to a Costar EIA/RIA 96well microtiter dish (Costar Corp, Cambridge Mass., USA) in 50 μLovernight at room temperature. The wells were washed three times with200 μL of PBS and the unbound sites blocked by the addition of 1% BSA inPBS (200 μl/well) for 1 hour at room temperature. The wells were washedas above. Biotinylated B7-1Ig or B7-2Ig (1 μg/ml serially diluted intwofold steps to 15.6 ng/mL; 50 μL) was added to each well and incubatedfor 2.5 hours at room temperature. The wells were washed as above. Thebound biotinylated B7-Ig was detected by the addition of 50 μl/well of a1:2000 dilution of streptavidin-HRP (Pierce Chemical Co., Rockford,Ill.) for 30 minutes at room temperature. The wells were washed as aboveand 50 μL of ABTS (Zymed, California) added and the developing bluecolor monitored at 405 nm after 30 min. The ability of unlabelled B7-1Igor B7-2Ig to compete with biotinylated B7-1Ig or B7-2Ig, respectively,was assessed by mixing varying amounts of the competing protein with aquantity of biotinylated B7-1Ig or B7-2Ig shown to be non-saturating(i.e., 70 ng/mL; 1.5 nM) and performing the binding assays as describedabove. A reduction in the signal (Abs 405 nm) expected for biotinylatedB17-1Ig or B7-2Ig indicated a competition for binding to immobilizedCTLA4Ig.

[0296] Considering the previous evidence that CTLA4 was the highaffinity receptor for B7-1, the avidity of binding of CTLA4 and CD28 toB7-1 and B7-2 was compared in assays as described above. In a firstexperiment, B7-1Ig or B7-2-Ig was labelled with biotin and bound toimmobilized CTLA4-Ig in the presence or absence of increasingconcentrations of unlabeled B7-1Ig or B7-2Ig. The experiment wasrepeated with ¹²⁵-I-labeled B7-1Ig or B7-2Ig. Representative results areshown in FIG. 16 (Panel A: B7-1Ig; Panel B: B7-2Ig). Using this solidphase binding assay, the avidity of B7-2 (2.7 nM) for CTLA4 wasdetermined to be approximately two-fold lower than that observed forB7-1 (4.6 nM). The experimentally determined IC₅₀ values are indicatedin the upper right comer of the panels. The affinity of both B7-1 andB7-2 for CD28 was lower and was difficult to confidently determine.

[0297] G. Direct Binding Assays of Modified Forms of B7 Family Membersto CTLA4Ig

[0298] Direct binding ELISA assays were performed to determine the levelof binding of B7 family members as Ig fusions proteins to CTLA4. Forthese assays, the immunoglobulin fusion proteins were attached to platesand the amount of biotinylated CTLA4 binding to the plates wasdetermined as described below.

[0299] Nunc Maxisorp plates were coated overnight at room temperaturewith 50 μl per well of a 20 μg/ml stock of the various B7Ig fusionproteins, or purified human IgG (Zymed) in PBS as described above. HumanCTLA4Ig (Repligen) was biotinylated using NHS LC biotin (Pierce,Rockford, Ill.). Varying amounts of biotinylated CTLA4Ig were added tothe plates and incubated for 2 hours at room temperature. The plateswere washed five times with PBS and then a 1:1000 dilution ofstreptaviden-HRP (Zymed) was added and left for 30′ minutes on theplates. After another series of washes with PBS, the HRP reactivity wasmeasured using an ABTS kit (Zymed) as described above.

[0300] The results, presented in FIG. 20, show that half-saturationoccurred at 500 pM for B7-1Ig, and at 5 nM and 8 nM for B7-2VIg andB7-2Ig, respectively Thus, CTLA4Ig binds to a similar extent to theB7-2VIg and B7-2Ig fusion proteins. CTLA4 does not, however bind toB7-2CIg. Thus, the variable domain of B7-2 is sufficient for binding toCTLA4.

[0301] H. Binding of B7-2VIg, B7-2Ig, and B7-1Ig to CHO-CTLA4 Cells

[0302] The examples presented in section F and G of Example 7 showedthat B7-2VIg and B7-2Ig bind soluble CTLA4Ig. The present example showsthat B7-2VIg and B7-2Ig also bind to CTLA4 expressed on a cell.

[0303] For this example, labeled B7-1Ig and B7-2Ig fusion proteins wereincubated with CHO cells transfected to express CTLA4 and binding wasmeasured by flow cytometry as follows.

[0304] B7-1 and B7-2 immunoglobulin fusion proteins, prepared asdescribed above, were diluted to 20 μig/ml in PBS/1%BSA and incubatedwith 10⁶ CHO cells transfected to express CTLA4 on their surface(CHO/CTLA4 cells) for 30 minutes on ice. The cells were washed twicewith cold PBS/BSA and incubated with a 1:50 dilution of goat anti-humanIgG-FITC (Zymed) for 30 minutes on ice. The cells were washed once withcold PBS/BSA, once with cold PBS and then resuspended in 250 μl coldPBS. The cells were then fixed by adding 250 μl of a 2% paraformaldehydesolution in PBS and incubation for at least 1 hour and the fluorescenceanalyzed using a FACS (Becton Dickinson, San Jose, Calif.). Similarlytreated CHO/CTLA4 cells which recieved the secondary antibody aloneserved to measure background staining.

[0305] The results of the flow cytometric analysis are presented in FIG.21. The results show that hB7-2Ig and hB7-2VIg fusion proteins bind to asimilar extent to CTLA4 positive cells (FIG. 21, panels C and D,repectively) and that the binding is stronger than binding of hB7-1Ig toCHO/CTLA4 cells (panel E).

[0306] I. B7-2VIg Binds with Stronger Affinity to CD28 than B7-1Ig andB7-2Ig

[0307] The Example shown in the previous section showed that B7-2VIg andB7-2Ig fusion proteins bind with similar affinity to cell membrane boundCTLA4. This example shows that the fusion proteins bind with differentaffinities to CD28 and in particular, that B7-2VIg binds with higheraffinity to CD28 than B7-2Ig.

[0308] B7-1 and B7-2 immunoglobulin fusion proteins diluted at 20 μg/mlin PBS/1%BSA were incubated with 10⁶ CHO cells transfected to expressCD28 on their surface (CHO/CD28 cells) for 30 minutes on ice. The cellswere washed twice with cold PBS/BSA and incubated with a 1:50 dilutionof goat anti-human IgG-FITC (Zymed) for 30 minutes on ice. The cellswere washed once with cold PBS/BSA, once with cold PBS and thenresuspended in 250 μl cold PBS. The cells were then fixed by adding 250μl of a 2% paraformaldehyde solution in PBS and incubation for at least1 hour and the fluorescence analyzed using a FACS (Becton Dickinson, SanJose, Calif.).

[0309] Representative results, as presented in FIG. 22, indicate thatB7-2Ig and B7-2VIg fusion proteins bind specifically to CHO-CD28 cells.The results further indicate that B7-2VIg protein binds to CD28 withstronger affinity than does B7-2Ig and B7-1Ig.

[0310] Thus, B7-2VIg fusion protein binds with higher affinity thanB⁷-2Ig to CD28, whereas both fusion proteins bind with similar affinityto CTLA4.

[0311] J. B7-2VIg is More Potent than B7-2Ig and B7-1Ig at CostimulatingProliferation of CD28+ T Cells

[0312] Since B7-2VIg binds with higher affinity to CD28 than B7-2Ig, itwas next investigated whether B7-2VIg fusion protein is also more potentat stimulating T cell proliferation than B7-2Ig fusion protein.

[0313] CD28+ T cells were isolated from peripheral blood leukocytes(PBLs) as described above. For measuring T cell proliferation, 1.2×10⁵CD28+ T cells were incubated in 200 μl of culture media in 96 wellplates and stimulated with PMA at 1 ng/ml and either of the followingcostimulatory signals: 6×10⁴ CHO/B7-1 or CHO/B7-2 cells (pretreatedovernight with mitomycin C and then extensively washed), 30 or 100 μg/mlof B7-1Ig, B7-2Ig, or B7-2VIg. Alternatively, the fusion proteins canfirst be incubated with a 3 fold excess (w/w) of affinity purified goatanti-human IgGFc (Cappel) for 30 minutes prior to use. After 60 hours ofincubation, the T cells were pulsed overnight with ³H-thymidine(Dupont/NEN) and harvested for counting, as described above.

[0314] The results of the proliferation assay are representedgraphically in FIG. 23. The results indicate that CHO expressed B7-1 andB7-2 strongly induced T cell proliferation. Purified B7-1Ig and B7-2Igalso induced proliferation, although not as potently as the CHO/B7-1 andCHO/B7-2 cells. However, B7-2VIg induced proliferation to the sameextent as the CHO/B7-1 and CHO/B7-2 cells. Thus, B7-2VIg is as potent asCHO/B7-1 and CHO/B7-2 cells in costimulating proliferation of T cells.

[0315] It another example, CD28+ T cells were activated with anti-CD3coated plates prepared as described above and costimulated with variousamounts of B7-1Ig, B7-2Ig , and B7-2VIg. Proliferation of the CD28+ Tcells was measured after 60 hours and overnight pulsing of the cellswith ³H-thymidine. The results, presented graphically in FIG. 24indicate that B7-2VIg fusion protein is also more potent than B7-2Ig andB7-1Ig at costimulating CD28+ T cells when anti-CD3 is used as theprimary activating agent. Moreover, the results indicate that B7-2VIg ismore potent than B7-2Ig and B7-1Ig at costimulating proliferation ofCD28+ T cells when low concentrations of the proteins are used. This isapparent when comparing the amount of thymidine incorporated in cellsincubated with 1 μg/ml of costimulatory fusion protein. In addition,B7-2VIg costimulates proliferation of the T cells at doses as low as 10ng/ml (250 pM).

[0316] Thus, the higher binding affinity of B7-2VIg versus B7-2Ig fusionprotein for CD28 (Example 7, section I) correlates with a highercostimulatory activity of B7-2VIg versus B7-2Ig for proliferatin of theT cells.

[0317] K. B7-2VIg is more Potent than B7-1Ig and B7-2Ig at CostimulatingProduction of IL-2 by CD28+ T Cells

[0318] It was next investigated whether B7-2VIg was also more potentthan B7-1Ig and B7-2Ig at costimulating activated T cells for theproduction of IL-2.

[0319] In the second example described in the previous section (SectionJ of Example 7), in which CD28+ T cells were activated with anti-CD3 andcostimulated with various amounts of B7-1Ig, B7-2Ig, and B7-2VIg, andcell proliferation measured, the level of IL-2 in the supernatant wasdetermined after 18 hours of stimulation using an ELISA kit (Endogen,Cambridge, Mass.). The results, presented in FIG. 24 show that more IL-2is produced by T cells costimulated with B7-2VIg than by T cellscostimulated with B7-1Ig or B7-2Ig. This was most apparent at lowconcentrations of costimulatory proteins (for example at 1 μg/ml of thefusion proteins).

[0320] Another example was performed to compare production of IL-2 fromCD28+ T cells costimulated with CHO/B7-2 cells or costimulated withB7-2VIg protein. CD28+ T cells were incubated in anti-CD3 coated platesin the presence of CHO/B7-2 cells or B7-2VIg protein for 1, 2, or 3days, and the amount of IL-2 was measured in the supernatant. Theresults, presented in FIG. 25, show that B7-2VIg is more potent atcostimulating T cells for the production of IL-2 than CHO/B7-2 cells.

[0321] In a further example, the amount of IL-2 produced by CD28+ Tcells costimulated with anti-CD28, B7-1Ig, B7-2Ig, or B7-2VIg wascompared after 1, 2, or 5 days of costimulation. CD28+ T cells wereactivated and costimulated with anti-CD28 antibody, or with B7-1Ig,B7-2Ig, or B7-2VIg fusion protein. The amount of IL-2 in the supernatantwas measured after 1, 2, and 5 days of costimulation. FIG. 26,representing graphically the amount of IL-2 produced by the T cells,indicate that B7-2IVIg is more potent than B7-2Ig and B7-1Ig atstimulating production of IL-2 by CD28+ T cells and further, that after5 days of culture, only T cells costimulated with B7-2Ig or B7-2VIgfusion proteins were producing IL-2. Moreover, T cells costimulated withB7-2VIg produce more IL-2 than T cells costimulated with B7-2Ig after 5days of culture.

[0322] Thus, B7-2VIg costimulate T cells to produce high levels of IL-2,even after at least 5 days of culture.

[0323] L. B7-2VIg is a Potent Costimulator of Activated CD4+ T Cells forthe Production of Cytokines

[0324] In this example, the amount of IL-2, IL-4, IFN-γ, and GM-CSFproduced by T cells costimulated with B7-2VIg, B7-2Ig, or B7-1Ig wascompared.

[0325] CD4+CD28+ T cells were cultured at 2×10⁶ cells/ml in T75 flaskscoated with anti-CD3 antibody alone, or with anti-CD28 mAb 9.3, or oneof the fusion proteins B7-1Ig, B7-2Ig, or B7-2VIg. After 18 hours ofculture, the amount of IL-2, IL-4, interferon-γ(IFN-γ), and granulocytemacrophage-colony stimulating factor (GM-CSF) was measured by ELISAusing commercially available kits (IL-2 (BioSource, Camarillo, Calif.),IL-4 (Endogen, Cambridge, Mass.), IFN-γ (Bio-Source, Camarillo, Calif.),and GM-CSF (R&D Systems, Minneapolis, Minn.)). The amount of IL-2 andIL-4 was also measured in cell costimulated for 120 hours.

[0326] The results are presented in Table VI. The results indicate thatcostimulation with B7-2VIg leads to production of high levels of IL-2,IL-4, IFN-γ, and GM-CSF. Moreover, the amount of all 4 cytokinesproduced from T cells costimulated with B7-2VIg was higher than theamount of cytokines produced from T cells costimulated with B7-1Ig.Compared to B7-2Ig, B7-2VIg also costimulated the production of higheramounts of IL-2, IL-4 and GM-CSF and similar amounts of IFN-γ. Thus,B7-2VIg is a potent costimulator for production of cytokines by T cells.

[0327] Moreover, after 120 hours of culture, the T cells costimulatedwith B7-2VIg produced more than twice the amount of IL-4 produced by Tcells costimulated with B7-2Ig and approximately 8 fold the amount ofIL-4 produced by T cells costimulated with B7-1 Ig. Thus, T cellscostimulated with B7-2VIg fusion protein induces production of highlevels of IL-4 and this production is longlasting. Costimulation of Tcells with B7-2VIg could thus drive T cells to a T helper 2 (Th2) statein long term culture. TABLE VI Cytokine production by CD4 + CD28 + Tcells costimulated with B7-2VIg, B7-2Ig, B7-1Ig or anti-CD28 antibodyIL-2 IL-2 IL-4 IL-4 IFN-g GM-CSF costimulus 18 hr 120 hr 18 hr 120 hr 18hr 18 hr none  19  0  6.5 0 27.6  0 anti-CD28 2412 N.D. 83.2 0 28   25B7-1Ig  734  0 10.4 5.7 27.8 10 B7-2Ig 1557 104 10.8 18.6 42.6 12B7-2vIg 3073 262 29.8 40.8 37.5 30

[0328] M. B7-2Ig and B7-2VIg. but not B7-1Ig Promote Sustained T CellGrowth

[0329] In this example, the capability of B7Ig fusion proteins topromote growth of T cells for extended periods was analysed.

[0330] CD28+ T lymphocytes were incubated in the presence of anti-CD3plus anti-CD28, or B7-1Ig, B7-2Ig, or B7-2VIg immobilized on beads andthe total cell numbers monitored over a period of 12 days. The resultsare presented in FIG. 27. Cells stimulated with anti-CD3 alone fail toproliferate, and die. Cells stimulated with anti-CD3 plus B7-2Ig gothrough one cycle of replication and then apoptose. Cells stimulatedwith anti-CD3 plus anti-CD28 or B7-2Ig or B7-2vIg continue to replicate.Thus, B7-2Ig and B7-2VIg fusion proteins, but not B7-1Ig, are capable ofstimulating prolonged growth of CD28+ T cells.

EXAMPLE 8 Production and Characterization of Monoclonal Antibodies toHuman B7-2

[0331] A. Immunizations and Cell Fusions

[0332] Balb/c female mice (obtained from Taconic Labs, Germantown, N.Y.)were immunized intraperitoneally with 50 μg human B7.2-Ig emulsified incomplete Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.) or 10⁶CHO-human B7.2 cells per mouse. The mice were given two boosterimmunizations with 10-25 μg human B7.2-Ig emulsified in incompleteFreund's adjuvant (Sigma Chemical Co., St. Louis, Mo.) or CHO-human B7.2cells at fourteen day intervals following the initial immunization forthe next two months. The mice were bled by retro-orbital bleed and thesera assayed for the presence of antibodies reactive to the immunogen byELISA against human B7.2-Ig. ELISA against hCTLA4-Ig was also used tocontrol for Ig tail directed antibody responses. Mice showing a strongserological response were boosted intravenously via the tail vein with25 μg human hB7.2-Ig diluted in phosphate-buffered saline (PBS), pH 7.2(GIBCO, Grand Island, N.Y.). Three to four days following this boost,the spleens from these mice were fused 5:1 with SP 2/0 myeloma cells(American Type Culture Collection, Rockville, Md., No. CRL8006), whichare incapable of secreting both heavy and light immunoglobulin chains(Kearney et al. (1979) J. Immunol. 123:1548). Standard methods basedupon those developed by Kohler and Milstein (Nature (1975) 256:495) wereused.

[0333] B. Antibody Screening

[0334] After 10-21 days, supernatants from wells containing hybridomacolonies from the fusion were screened for the presence of antibodiesreactive to human B7.2 as follows: Each well of a 96 well flat bottomedplate (Costar Corp., Cat. #3590) was coated with 50 μl per well of a 1μg/ml human B7.2-Ig solution or 5×10⁴ 3T3-hB7.2 cells on lysine coatedplates in phosphate-buffered saline, pH 7.2, overnight at 4° C. Thehuman B7.2-Ig solution was aspirated off, or the cells were cross-linkedto the plates with glutaraldehyde, and the wells were washed three timeswith PBS, then blocked with 1% BSA solution (in PBS) (100 μl/well) forone hour at room temperature. Following this blocking incubation, thewells were washed three times with PBS and 50 μl of hybridomasupernatant was added per well and incubated for 1.5 hours at roomtemperature. Following this incubation, the wells were washed threetimes with PBS and then incubated for 1.5 hours at room temperature with50 μl per well of a 1:4000 dilution of horseradishperoxidase-conjugated, affinity purified, goat anti-mouse IgG or IgMheavy and light chain-specific antibodies (HRP; Zymed Laboratories, SanFrancisco, Calif.). The wells were then washed three times with PBS,followed by a 30 minute incubation in 50 μl per well of 1 mM2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) in 0.1 MNa-Citrate, pH 4.2 to which a 1:1000 dilution of 30% hydrogen peroxidehad been added as a substrate for HRP to detect bound antibody. Theabsorbence was then determined at OD₄₁₀ on a spectrophotometricautoreader (Dynatech, Virginia).

[0335] Three hybridomas, HA3.1F9, HA5.2B7 and HF2.3D1, were identifiedthat produced antibodies to human B7.2-Ig. HA3.1 F9 was determined to beof the IgG1 isotype, HA5.2B7 was determined to be of the IgG2b isotypeand HF2.3D1 as determined to be of the IgG2a isotype. Each of thesehybridomas were subcloned two additional times to insure that they weremonoclonal. Hybidoma cells were deposited with the American Type CultureCollection, which meets the requirements of the Budapest Treaty, on Jul.19, 1994 as ATCC Accession No. (hybridoma HA3.1F9), ATCC Accession No.(HA5.2B7) and ATCC Accession No. _ (HF2.3D1).

[0336] C. Competitive ELISA

[0337] Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1were further characterized by competitive ELISA, in which the ability ofthe monoclonal antibodies to inhibit the binding of biotinylatedhCTLA4Ig to immobilized hB7-2 immunoglobulin fusion proteins wasexamined. Biotinylation of hCTLA4Ig was performed using PierceImmunopure NHS-LC Biotin (Cat. No. 21335). B7-2 immunoglobulin fusionproteins used were: hB7.2-Ig (full-length hB7-2), hB7.2-VIg (hB7-2variable domain only) and hB7.2-CIg (B7-2 constant domain only). AhB7.1-Ig fusion protein was used as a control. For the ELISA, 96 wellplates were coated with the Ig fusion protein (50 μl/well of a 20 μg/mlsolution) overnight at room temperature. The wells were washed threetimes with PBS, blocked with 10% fetal bovine serum (FBS), 0.1% bovineserum albumin (BSA) in PBS for 1 hour at room temperature, and washedagain three times with PBS. To each well was added 50 μl ofBio-hCTLA4-Ig (70 ng/ml) and 50 μl of competitor monoclonal antibodysupernatant. Control antibodies were an anti-B7.1 mAb (EW3.5D12) and theanti-hB7-2 mAb B70 (IgG2bκ, obtained from Pharmingen). The wells werewashed again and streptavidin-conjugated horse radish peroxidase (fromPierce, Cat. No. 21126; 1:2000 dilution, 50 μl/well) was added andincubated for 30 minutes at room temperature. The wells were washedagain, followed by a 30 minute incubation in 50 μl per well of ABTS in0.1 M Na-Citrate, pH 4.2 to which a 1:1000 dilution of 30% hydrogenperoxide had been added as a substrate for HRP to detect bound antibody.The absorbence was then determined at OD₄₁₀ on a spectrophotometricautoreader (Dynatech, Virginia). The results, shown in Table IV below,demonstrate that each of the mAbs produced by the hybridomas HA3.1F9,HA5.2B7 and HF2.3D1 are able to competitively inhibit the binding ofhCLTA4Ig to full-length hB7.2-Ig or hB7.2-VIg (hCTLA4Ig does not bind tohB7.2CIg). TABLE IV Blocking of Binding hB7.1-Ig hB7.2-Ig hB7.2-VIghB7.2-CIg EW3.5D12 Yes No No No (anti-hB7.1 mAb) B70 (anti-hB7-2) No YesYes No HA3.1F9 (anti-hB7-2) No Yes Yes No HA5.2B7 (anti-hB7-2) No YesYes No HF2.3D1 (anti-hB7-2) No Yes Yes No

[0338] D. Flow Cytometry

[0339] Supernatants from the hybridomas HA3.1F9, HA5.2B7 and HF2.3D1were also characterized by flow cytometry. Supernatants collected fromthe clones were screened by flow cytometry on CHO and 3T3 cellstransfected to express hB7.2 (CHO-hB7.2 and 3T3-hB7.2, respectively) orcontrol transfected 3T3 cells (3T3-Neo). Flow cytometry was performed asfollows: 1×10⁶ cells were washed three times in 1% BSA in PBS, then thecells were incubated in 50 μl hybridoma supernatant or culture media per1×10⁶ cells for 30 minutes at 4° C. Following the incubation, the cellswere washed three times with 1% BSA in PBS, then incubated in 50 μlfluorescein-conjugated goat anti-mouse IgG or IgM antibodies (ZymedLaboratories, San Francisco, Calif.) at 1:50 dilution per 1×10⁶ cellsfor 30 minutes at 4° C. The cells were then washed three times in 1% BSAin PBS and fixed with 1% paraformaldehyde solution. The cell sampleswere then analyzed on a FACScan flow cytometer (Becton Dickinson, SanJose Calif.). The results, shown in FIGS. 17, 18 and 19, demonstrate themonoclonal antibodies produced by the hybridomas HA3.1F9, HA5.2B7 andHF2.3D1 each bind to hB7-2 on the surface of cells.

[0340] E. Inhibition of Proliferation of Human T Cells by Anti-hB7-2mAbs

[0341] Hybridoma supernatants containing anti-human B7-2 mAbs weretested for their ability to inhibit hB7-2 costimulation of human Tcells. In this assay, purified CD28⁺ human T cells were treated withsubmitogenic amounts of PMA (1 ng/ml) to deliver the primary signal andwith CHO cells expressing hB7-2 on their surface to deliver thecostimulatory signal. Proliferation of the T cells was measured afterthree days in culture by the addition of ³H-thymidine for the remaining18 hours. As shown in Table V, resting T cells show little proliferationas measured by ³H-thymidine incorporation (510 pm). Delivery of signal 1by PMA results in some proliferation (3800 pm) and T cells receivingboth the primary (PMA) and costimulatory (CHO/hB7-2) signals proliferatemaximally (9020 cpm). All three anti-hB7-2 mAbs tested reduce thecostimulatory signal induced proliferation to that found for PMA treatedcells alone showing that these mAbs can inhibit T cell proliferation byblocking the B7/CD28 costimulatory pathway. TABLE V Addition to CD28⁺ TCells hB7-2 mAb CPM — —  510 +PMA — 3800 +PMA + CHO/hB7-2 — 9020 +PMA +CHO/hB7-2 HF2.301 3030 — HA5.2B7 1460 — HA3.1F9 2980

[0342] F. Antibodies to the B7-2 Variable Domain Block B7-2 Function

[0343] In this example, the ability of a series of monoclonal antibodiesto B7-2 to bind to the Ig-variable or Ig-constant domains of B7-2, an toinhibit T cell proliferation was analyzed.

[0344] Monoclonal antibodies to human B7-1 and B7-2 were prepared fromBalb/c mice using SP2/0 cells and standard protocols. Briefly, Balb/cfemale mice (Taconic Labs, Germantown, N.Y.) were immunizedintraperitoneally with either 50 μgs B7-2Ig emulsified in CFA (Sigma,St. Louis, Mo.) or 10⁶ CHO/B7-2 cells. The mice were boosted twice at 14day intervals following the initial immunization and once with B7-2Igprotein in PBS. Hybridoma colonies were established in 96 well tissueculture plates and the culture supernatants were assayed for directbinding to B7-2Ig. All mAbs were purified from ascites fluid onProtein-A sepharose as described above. MAb B70 was purchased fromPharMingen (San Diego, Calif.).

[0345] Purified mAbs were tested for their ability to bind to thevarious B7-Ig forms as follows. Maxisorp plates (Nunc) were coatedovernight at room temperature with 20 μg/ml of purified B7-2Ig proteinin PBS. The plates were then blocked with PBS/0.1%BSA for 1 h. Purifiedantibody (5 μgs/ml in PBS) was added to the test wells, the platesincubated for 1 hour, and then washed 5 times with PBS/0.05% Tween20.Goat anti-mouse IgG-HRP (Zymed) was added and allowed to react for 1hour, followed by 5 washes. The plates were developed as describedabove.

[0346] The binding characteristics of the antibodies is indicated inTable VII under the heading “Recognition”. As indicated in Table VII,all antibodies recognized B7-2Ig. Binding of the antibodies to B7-2VIgand B7-2CIg fusion proteins indicated that the antibodies recognizeeither the variable region construct or the constant region construct,but not both constructs.

[0347] The antibodies were further analyzed for their ability to inhibitbinding of B7-2Ig to CTLA4 and to CD28 and to inhibit T cellproliferation. Using a competition ELISA format, varying amounts of mAbswere added to wells coated B7-2Ig and containing 35 ngs/ml biotinylatedCTLA4Ig or CD28Ig. The ability of mAbs to disrupt the binding wasmeasured as a decrease in the specific signal of captured biotinylatedCTLA4Ig or CD28Ig. The capability of the antibodies to inhibitproliferation of T cells was determined by performing proliferationassays, as described above in which one of the antibodies was added.

[0348] The results are presented in Table VII under the heading“Inhibition”. In general, antibodies to the V-domain of B7-2 inhibitbinding of the B7-2Ig to CD28 and CTLA4 and also inhibit CHO/B7-2 drivenT cell proliferation. Antibodies to the C-domain are not inhibitorysuggesting that the functionality of B7-2 resides in the V-domain. TABLEVII Characterization of anti-B7-2 antibodies Recognition Inhibition B7vB7c CTLA4 CD28 T cell Anti-B7-2 mAbs B7 domain domain binding bindingprolif. HA5.1F9 IgG1 + − − + + + HA5.2B7 IgG2b + + − + + + HF2.3D11IgG2a + + − + + + HF4.3C11 IgG1 + + − + + + HF4.3E8 IgG1 + + − + +/− +/−HF4.5B4 IgG1 + − + nd − − HF4.5H12 IgM + − + − − − HF4.6B1 IgG2a + − + −− − HF4.6H4 IgG1 + + − + + + B70 IgG2b + + − + + +

[0349] Moreover, all of the anti-B7-1 and anti-B7-2 mAbs tested alsorecognized their respective ligand when expressed on the surface of CHOcells and on the surface of activated human B cells where tested. Noneof the anti-B7-1 or anti-B7-2 mAbs showed any crossreactivity with theother B7 protein.

[0350] Equivalents

[0351] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:55 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1120 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 107..1093 (xi) SEQUENCE DESCRIPTION: SEQ IDNO:1: CACAGGGTGA AAGCTTTGCT TCTCTGCTGC TGTAACAGGG ACTAGCACAG ACACACGGAT60 GAGTGGGGTC ATTTCCAGAT ATTAGGTCAC AGCAGAAGCA GCCAAA ATG GAT CCC 115Met Asp Pro 1 CAG TGC ACT ATG GGA CTG AGT AAC ATT CTC TTT GTG ATG GCCTTC CTG 163 Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met Ala PheLeu 5 10 15 CTC TCT GGT GCT GCT CCT CTG AAG ATT CAA GCT TAT TTC AAT GAGACT 211 Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr20 25 30 35 GCA GAC CTG CCA TGC CAA TTT GCA AAC TCT CAA AAC CAA AGC CTGAGT 259 Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser40 45 50 GAG CTA GTA GTA TTT TGG CAG GAC CAG GAA AAC TTG GTT CTG AAT GAG307 Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu 5560 65 GTA TAC TTA GGC AAA GAG AAA TTT GAC AGT GTT CAT TCC AAG TAT ATG355 Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met 7075 80 GGC CGC ACA AGT TTT GAT TCG GAC AGT TGG ACC CTG AGA CTT CAC AAT403 Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn 8590 95 CTT CAG ATC AAG GAC AAG GGC TTG TAT CAA TGT ATC ATC CAT CAC AAA451 Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys 100105 110 115 AAG CCC ACA GGA ATG ATT CGC ATC CAC CAG ATG AAT TCT GAA CTGTCA 499 Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser120 125 130 GTG CTT GCT AAC TTC AGT CAA CCT GAA ATA GTA CCA ATT TCT AATATA 547 Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile135 140 145 ACA GAA AAT GTG TAC ATA AAT TTG ACC TGC TCA TCT ATA CAC GGTTAC 595 Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr150 155 160 CCA GAA CCT AAG AAG ATG AGT GTT TTG CTA AGA ACC AAG AAT TCAACT 643 Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr165 170 175 ATC GAG TAT GAT GGT ATT ATG CAG AAA TCT CAA GAT AAT GTC ACAGAA 691 Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu180 185 190 195 CTG TAC GAC GTT TCC ATC AGC TTG TCT GTT TCA TTC CCT GATGTT ACG 739 Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp ValThr 200 205 210 AGC AAT ATG ACC ATC TTC TGT ATT CTG GAA ACT GAC AAG ACGCGG CTT 787 Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr ArgLeu 215 220 225 TTA TCT TCA CCT TTC TCT ATA GAG CTT GAG GAC CCT CAG CCTCCC CCA 835 Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro ProPro 230 235 240 GAC CAC ATT CCT TGG ATT ACA GCT GTA CTT CCA ACA GTT ATTATA TGT 883 Asp His Ile Pro Trp Ile Thr Ala Val Leu Pro Thr Val Ile IleCys 245 250 255 GTG ATG GTT TTC TGT CTA ATT CTA TGG AAA TGG AAG AAG AAGAAG CGG 931 Val Met Val Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys Lys LysArg 260 265 270 275 CCT CGC AAC TCT TAT AAA TGT GGA ACC AAC ACA ATG GAGAGG GAA GAG 979 Pro Arg Asn Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu ArgGlu Glu 280 285 290 AGT GAA CAG ACC AAG AAA AGA GAA AAA ATC CAT ATA CCTGAA AGA TCT 1027 Ser Glu Gln Thr Lys Lys Arg Glu Lys Ile His Ile Pro GluArg Ser 295 300 305 GAT GAA GCC CAG CGT GTT TTT AAA AGT TCG AAG ACA TCTTCA TGC GAC 1075 Asp Glu Ala Gln Arg Val Phe Lys Ser Ser Lys Thr Ser SerCys Asp 310 315 320 AAA AGT GAT ACA TGT TTT TAATTAAAGA GTAAAGCCCAAAAAAAA 1120 Lys Ser Asp Thr Cys Phe 325 (2) INFORMATION FOR SEQ IDNO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 329 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Pro Gln Cys Thr Met Gly LeuSer Asn Ile Leu Phe Val Met 1 5 10 15 Ala Phe Leu Leu Ser Gly Ala AlaPro Leu Lys Ile Gln Ala Tyr Phe 20 25 30 Asn Glu Thr Ala Asp Leu Pro CysGln Phe Ala Asn Ser Gln Asn Gln 35 40 45 Ser Leu Ser Glu Leu Val Val PheTrp Gln Asp Gln Glu Asn Leu Val 50 55 60 Leu Asn Glu Val Tyr Leu Gly LysGlu Lys Phe Asp Ser Val His Ser 65 70 75 80 Lys Tyr Met Gly Arg Thr SerPhe Asp Ser Asp Ser Trp Thr Leu Arg 85 90 95 Leu His Asn Leu Gln Ile LysAsp Lys Gly Leu Tyr Gln Cys Ile Ile 100 105 110 His His Lys Lys Pro ThrGly Met Ile Arg Ile His Gln Met Asn Ser 115 120 125 Glu Leu Ser Val LeuAla Asn Phe Ser Gln Pro Glu Ile Val Pro Ile 130 135 140 Ser Asn Ile ThrGlu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile 145 150 155 160 His GlyTyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys 165 170 175 AsnSer Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn 180 185 190Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro 195 200205 Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys 210215 220 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln225 230 235 240 Pro Pro Pro Asp His Ile Pro Trp Ile Thr Ala Val Leu ProThr Val 245 250 255 Ile Ile Cys Val Met Val Phe Cys Leu Ile Leu Trp LysTrp Lys Lys 260 265 270 Lys Lys Arg Pro Arg Asn Ser Tyr Lys Cys Gly ThrAsn Thr Met Glu 275 280 285 Arg Glu Glu Ser Glu Gln Thr Lys Lys Arg GluLys Ile His Ile Pro 290 295 300 Glu Arg Ser Asp Glu Ala Gln Arg Val PheLys Ser Ser Lys Thr Ser 305 310 315 320 Ser Cys Asp Lys Ser Asp Thr CysPhe 325 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi)SEQUENCE DESCRIPTION: SEQ ID NO:3: TAATACGACT CACTATAGGG 20 (2)INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:4: TAAGGTTCCT TCACAAAG 18 (2) INFORMATION FOR SEQID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:ACTGGTAGGT ATGGAAGATC C 21 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGCGAATCATTCCTGTGGG C 21 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AAAGCCCACAGGAATGATTC G 21 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CTCTCAAAACCAAAGCCTGA G 21 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTAGGTCACAGCAGAAGCAG C 21 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TCTGGAAACTGACAAGACGC G 21 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CTCAGGCTTTGGTTTTGAGA G 21 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CACTCTCTTCCCTCTCCATT G 21 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GACAAGCTGATGGAAACGTC G 21 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: CAATGGAGAGGGAAGAGAGT G 21 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CTTTAGAGCA CA12 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 8 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:16: CTCTAAAG 8 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:17: Lys Tyr Met Gly Arg Thr Ser Phe Asp 5 (2) INFORMATION FORSEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Lys Ser Gln Asp Asn Val Thr GluLys Tyr Asp Val Ser 5 10 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:19: Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn Ser Tyr LysCys 5 10 15 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TGGCCCATGGCTTCAGA 17 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCCAAAATGGATCCCCA 17 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1163 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 111..1040 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:22: CCCACGCGTC CGGGAGCAAG CAGACGCGTA AGAGTGGCTCCTGTAGGCAG CACGGACTTG 60 AACAACCAGA CTCCTGTAGA CGTGTTCCAG AACTTACGGAAGCACCCACG ATG GAC 116 Met Asp 1 CCC AGA TGC ACC ATG GGC TTG GCA ATC CTTATC TTT GTG ACA GTC TTG 164 Pro Arg Cys Thr Met Gly Leu Ala Ile Leu IlePhe Val Thr Val Leu 5 10 15 CTG ATC TCA GAT GCT GTT TCC GTG GAG ACG CAAGCT TAT TTC AAT GGG 212 Leu Ile Ser Asp Ala Val Ser Val Glu Thr Gln AlaTyr Phe Asn Gly 20 25 30 ACT GCA TAT CTG CCG TGC CCA TTT ACA AAG GCT CAAAAC ATA AGC CTG 260 Thr Ala Tyr Leu Pro Cys Pro Phe Thr Lys Ala Gln AsnIle Ser Leu 35 40 45 50 AGT GAG CTG GTA GTA TTT TGG CAG GAC CAG CAA AAGTTG GTT CTG TAC 308 Ser Glu Leu Val Val Phe Trp Gln Asp Gln Gln Lys LeuVal Leu Tyr 55 60 65 GAG CAC TAT TTG GGC ACA GAG AAA CTT GAT AGT GTG AATGCC AAG TAC 356 Glu His Tyr Leu Gly Thr Glu Lys Leu Asp Ser Val Asn AlaLys Tyr 70 75 80 CTG GGC CGC ACG AGC TTT GAC AGG AAC AAC TGG ACT CTA CGACTT CAC 404 Leu Gly Arg Thr Ser Phe Asp Arg Asn Asn Trp Thr Leu Arg LeuHis 85 90 95 AAT GTT CAG ATC AAG GAC ATG GGC TCG TAT GAT TGT TTT ATA CAAAAA 452 Asn Val Gln Ile Lys Asp Met Gly Ser Tyr Asp Cys Phe Ile Gln Lys100 105 110 AAG CCA CCC ACA GGA TCA ATT ATC CTC CAA CAG ACA TTA ACA GAACTG 500 Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gln Thr Leu Thr Glu Leu115 120 125 130 TCA GTG ATC GCC AAC TTC AGT GAA CCT GAA ATA AAA CTG GCTCAG AAT 548 Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Ile Lys Leu Ala GlnAsn 135 140 145 GTA ACA GGA AAT TCT GGC ATA AAT TTG ACC TGC ACG TCT AAGCAA GGT 596 Val Thr Gly Asn Ser Gly Ile Asn Leu Thr Cys Thr Ser Lys GlnGly 150 155 160 CAC CCG AAA CCT AAG AAG ATG TAT TTT CTG ATA ACT AAT TCAACT AAT 644 His Pro Lys Pro Lys Lys Met Tyr Phe Leu Ile Thr Asn Ser ThrAsn 165 170 175 GAG TAT GGT GAT AAC ATG CAG ATA TCA CAA GAT AAT GTC ACAGAA CTG 692 Glu Tyr Gly Asp Asn Met Gln Ile Ser Gln Asp Asn Val Thr GluLeu 180 185 190 TTC AGT ATC TCC AAC AGC CTC TCT CTT TCA TTC CCG GAT GGTGTG TGG 740 Phe Ser Ile Ser Asn Ser Leu Ser Leu Ser Phe Pro Asp Gly ValTrp 195 200 205 210 CAT ATG ACC GTT GTG TGT GTT CTG GAA ACG GAG TCA ATGAAG ATT TCC 788 His Met Thr Val Val Cys Val Leu Glu Thr Glu Ser Met LysIle Ser 215 220 225 TCC AAA CCT CTC AAT TTC ACT CAA GAG TTT CCA TCT CCTCAA ACG TAT 836 Ser Lys Pro Leu Asn Phe Thr Gln Glu Phe Pro Ser Pro GlnThr Tyr 230 235 240 TGG AAG GAG ATT ACA GCT TCA GTT ACT GTG GCC CTC CTCCTT GTG ATG 884 Trp Lys Glu Ile Thr Ala Ser Val Thr Val Ala Leu Leu LeuVal Met 245 250 255 CTG CTC ATC ATT GTA TGT CAC AAG AAG CCG AAT CAG CCTAGC AGG CCC 932 Leu Leu Ile Ile Val Cys His Lys Lys Pro Asn Gln Pro SerArg Pro 260 265 270 AGC AAC ACA GCC TCT AAG TTA GAG CGG GAT AGT AAC GCTGAC AGA GAG 980 Ser Asn Thr Ala Ser Lys Leu Glu Arg Asp Ser Asn Ala AspArg Glu 275 280 285 290 ACT ATC AAC CTG AAG GAA CTT GAA CCC CAA ATT GCTTCA GCA AAA CCA 1028 Thr Ile Asn Leu Lys Glu Leu Glu Pro Gln Ile Ala SerAla Lys Pro 295 300 305 AAT GCA GAG TGAAGGCAGT GAGAGCCTGA GGAAAGAGTTAAAAATTGCT 1077 Asn Ala Glu TTGCCTGAAA TAAGAAGTGC AGAGTTTCTC AGAATTCAAAAATGTTCTCA GCTGATTGGA 1137 ATTCTACAGT TGAATAATTA AAGAAC 1163 (2)INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:309 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Asp Pro ArgCys Thr Met Gly Leu Ala Ile Leu Ile Phe Val Thr 1 5 10 15 Val Leu LeuIle Ser Asp Ala Val Ser Val Glu Thr Gln Ala Tyr Phe 20 25 30 Asn Gly ThrAla Tyr Leu Pro Cys Pro Phe Thr Lys Ala Gln Asn Ile 35 40 45 Ser Leu SerGlu Leu Val Val Phe Trp Gln Asp Gln Gln Lys Leu Val 50 55 60 Leu Tyr GluHis Tyr Leu Gly Thr Glu Lys Leu Asp Ser Val Asn Ala 65 70 75 80 Lys TyrLeu Gly Arg Thr Ser Phe Asp Arg Asn Asn Trp Thr Leu Arg 85 90 95 Leu HisAsn Val Gln Ile Lys Asp Met Gly Ser Tyr Asp Cys Phe Ile 100 105 110 GlnLys Lys Pro Pro Thr Gly Ser Ile Ile Leu Gln Gln Thr Leu Thr 115 120 125Glu Leu Ser Val Ile Ala Asn Phe Ser Glu Pro Glu Ile Lys Leu Ala 130 135140 Gln Asn Val Thr Gly Asn Ser Gly Ile Asn Leu Thr Cys Thr Ser Lys 145150 155 160 Gln Gly His Pro Lys Pro Lys Lys Met Tyr Phe Leu Ile Thr AsnSer 165 170 175 Thr Asn Glu Tyr Gly Asp Asn Met Gln Ile Ser Gln Asp AsnVal Thr 180 185 190 Glu Leu Phe Ser Ile Ser Asn Ser Leu Ser Leu Ser PhePro Asp Gly 195 200 205 Val Trp His Met Thr Val Val Cys Val Leu Glu ThrGlu Ser Met Lys 210 215 220 Ile Ser Ser Lys Pro Leu Asn Phe Thr Gln GluPhe Pro Ser Pro Gln 225 230 235 240 Thr Tyr Trp Lys Glu Ile Thr Ala SerVal Thr Val Ala Leu Leu Leu 245 250 255 Val Met Leu Leu Ile Ile Val CysHis Lys Lys Pro Asn Gln Pro Ser 260 265 270 Arg Pro Ser Asn Thr Ala SerLys Leu Glu Arg Asp Ser Asn Ala Asp 275 280 285 Arg Glu Thr Ile Asn LeuLys Glu Leu Glu Pro Gln Ile Ala Ser Ala 290 295 300 Lys Pro Asn Ala Glu305 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:24: ACATAAGCCT GAGTGAGCTG G 21 (2) INFORMATIONFOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 basepairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION:SEQ ID NO:25: ATGATGAGCA GCATCACAAG G 21 (2) INFORMATION FOR SEQ IDNO:26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:TGGTCGAGTG AGTCCGAATA C 21 (2) INFORMATION FOR SEQ ID NO:27: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GACGAGTAGTAACATACAGT G 21 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 1491 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA tomRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: no (vi) ORIGINAL SOURCE:(A) ORGANISM: Homo sapien (F) TISSUE TYPE: lymphoid (G) CELL TYPE: Bcell (H) CELL LINE: Raji (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA inpCDM8 vector (B) CLONE: B7, Raji clone #13 (viii) POSITION IN GENOME:(A) CHROMOSOME/SEGMENT: 3 (ix) FEATURE: (A) NAME/KEY: Open reading frame(translated region) (B) LOCATION: 318 to 1181 bp (C) IDENTIFICATIONMETHOD: similarity to other pattern (ix) FEATURE: (A) NAME/KEY:Alternate polyadenylation signal (B) LOCATION: 1474 to 1479 bp (C)IDENTIFICATION METHOD: similarity to other pattern (x) PUBLICATIONINFORMATION: (A) AUTHORS: FREEMAN, GORDON J. FREEDMAN, ARNOLD S. SEGIL,JEFFREY M. LEE, GRACE WHITMAN, JAMES F. NADLER, LEE M. (B) TITLE: B7, ANew Member Of The Ig Superfamily With Unique Expression On Activated AndNeoplastic B Cells (C) JOURNAL: The Journal of Immunology (D) VOLUME:143 (E) ISSUE: 8 (F) PAGES: 2714-2722 (G) DATE: 15-OCT-1989 (H) RELEVANTRESIDUES IN SEQ ID NO:28: FROM 1 TO 1491 (xi) SEQUENCE DESCRIPTION: SEQID NO:28: CCAAAGAAAA AGTGATTTGT CATTGCTTTA TAGACTGTAA GAAGAGAACATCTCAGAAGT 60 GGAGTCTTAC CCTGAAATCA AAGGATTTAA AGAAAAAGTG GAATTTTTCTTCAGCAAGCT 120 GTGAAACTAA ATCCACAACC TTTGGAGACC CAGGAACACC CTCCAATCTCTGTGTGTTTT 180 GTAAACATCA CTGGAGGGTC TTCTACGTGA GCAATTGGAT TGTCATCAGCCCTGCCTGTT 240 TTGCACCTGG GAAGTGCCCT GGTCTTACTT GGGTCCAAAT TGTTGGCTTTCACTTTTGAC 300 CCTAAGCATC TGAAGCC ATG GGC CAC ACA CGG AGG CAG GGA ACATCA CCA TCC 353 Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser -30 -25AAG TGT CCA TAC CTG AAT TTC TTT CAG CTC TTG GTG CTG GCT GGT CTT 401 LysCys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu -20 -15 -10TCT CAC TTC TGT TCA GGT GTT ATC CAC GTG ACC AAG GAA GTG AAA GAA 449 SerHis Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu -5 1 5 10GTG GCA ACG CTG TCC TGT GGT CAC AAT GTT TCT GTT GAA GAG CTG GCA 497 ValAla Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala 15 20 25 CAAACT CGC ATC TAC TGG CAA AAG GAG AAG AAA ATG GTG CTG ACT ATG 545 Gln ThrArg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met 30 35 40 ATG TCTGGG GAC ATG AAT ATA TGG CCC GAG TAC AAG AAC CGG ACC ATC 593 Met Ser GlyAsp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile 45 50 55 TTT GAT ATCACT AAT AAC CTC TCC ATT GTG ATC CTG GCT CTG CGC CCA 641 Phe Asp Ile ThrAsn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro 60 65 70 TCT GAC GAG GGCACA TAC GAG TGT GTT GTT CTG AAG TAT GAA AAA GAC 689 Ser Asp Glu Gly ThrTyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp 75 80 85 90 GCT TTC AAG CGGGAA CAC CTG GCT GAA GTG ACG TTA TCA GTC AAA GCT 737 Ala Phe Lys Arg GluHis Leu Ala Glu Val Thr Leu Ser Val Lys Ala 95 100 105 GAC TTC CCT ACACCT AGT ATA TCT GAC TTT GAA ATT CCA ACT TCT AAT 785 Asp Phe Pro Thr ProSer Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn 110 115 120 ATT AGA AGG ATAATT TGC TCA ACC TCT GGA GGT TTT CCA GAG CCT CAC 833 Ile Arg Arg Ile IleCys Ser Thr Ser Gly Gly Phe Pro Glu Pro His 125 130 135 CTC TCC TGG TTGGAA AAT GGA GAA GAA TTA AAT GCC ATC AAC ACA ACA 881 Leu Ser Trp Leu GluAsn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr 140 145 150 GTT TCC CAA GATCCT GAA ACT GAG CTC TAT GCT GTT AGC AGC AAA CTG 929 Val Ser Gln Asp ProGlu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu 155 160 165 170 GAT TTC AATATG ACA ACC AAC CAC AGC TTC ATG TGT CTC ATC AAG TAT 977 Asp Phe Asn MetThr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr 175 180 185 GGA CAT TTAAGA GTG AAT CAG ACC TTC AAC TGG AAT ACA ACC AAG CAA 1025 Gly His Leu ArgVal Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln 190 195 200 GAG CAT TTTCCT GAT AAC CTG CTC CCA TCC TGG GCC ATT ACC TTA ATC 1073 Glu His Phe ProAsp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile 205 210 215 TCA GTA AATGGA ATT TTT GTG ATA TGC TGC CTG ACC TAC TGC TTT GCC 1121 Ser Val Asn GlyIle Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala 220 225 230 CCA AGA TGCAGA GAG AGA AGG AGG AAT GAG AGA TTG AGA AGG GAA AGT 1169 Pro Arg Cys ArgGlu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser 235 240 245 250 GTA CGCCCT GTA TAACAGTGTC CGCAGAAGCA AGGGGCTGAA AAGATCTGAA 1221 Val Arg Pro ValGGTAGCCTCC GTCATCTCTT CTGGGATACA TGGATCGTGG GGATCATGAG GCATTCTTCC 1281CTTAACAAAT TTAAGCTGTT TTACCCACTA CCTCACCTTC TTAAAAACCT CTTTCAGATT 1341AAGCTGAACA GTTACAAGAT GGCTGGCATC CCTCTCCTTT CTCCCCATAT GCAATTTGCT 1401TAATGTAACC TCTTCTTTTG CCATGTTTCC ATTCTGCCAT CTTGAATTGT CTTGTCAGCC 1461AATTCATTAT CTATTAAACA CTAATTTGAG 1491 (2) INFORMATION FOR SEQ ID NO:29:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 288 amino acids (B) TYPE:amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (A)DESCRIPTION: B cell activation antigen; natural ligand for CD28 T cellsurface antigen; transmembrane protein (ix) FEATURE: (A) NAME/KEY:signal sequence (B) LOCATION: -34 to -1 (C) IDENTIFICATION METHOD: aminoterminal sequencing of soluble protein (D) OTHER INFORMATION:hydrophobic (ix) FEATURE: (A) NAME/KEY: extracellular domain (B)LOCATION: 1 to 208 (C) IDENTIFICATION METHOD: similarity with knownsequence (ix) FEATURE: (A) NAME/KEY: transmembrane domain (B) LOCATION:209 to 235 (C) IDENTIFICATION METHOD: similarity with known sequence(ix) FEATURE: (A) NAME/KEY: intracellular domain (B) LOCATION: 236 to254 (C) IDENTIFICATION METHOD: similarity with known sequence (ix)FEATURE: (A) NAME/KEY: N-linked glycosylation (B) LOCATION: 19 to 21 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 55 to 57 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 64 to 66 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 152 to 154 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 173 to 175 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 177 to 179 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 192 to 194 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: N-linked glycosylation (B) LOCATION: 198 to 200 (C)IDENTIFICATION METHOD: similarity with known sequence (ix) FEATURE: (A)NAME/KEY: Ig V-set domain (B) LOCATION: 1 to 104 (C) IDENTIFICATIONMETHOD: similarity with known sequence (ix) FEATURE: (A) NAME/KEY: IgC-set domain (B) LOCATION: 105 to 202 (C) IDENTIFICATION METHOD:similarity with known sequence (x) PUBLICATION INFORMATION: (A) AUTHORS:FREEMAN, GORDON J. FREEDMAN, ARNOLD S. SEGIL, JEFFREY M. LEE, GRACEWHITMAN, JAMES F. NADLER, LEE M. (B) TITLE: B7, A New Member Of The IgSuperfamily With Unique Expression On Activated And Neoplastic B Cells(C) JOURNAL: The Journal of Immunology (D) VOLUME: 143 (E) ISSUE: 8 (F)PAGES: 2714-2722 (G) DATE: 15-OCT-1989 (H) RELEVANT RESIDUES IN SEQ IDNO:29: From -26 to 262 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Met GlyHis Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr -30 -25 -20 LeuAsn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys -15 -10 -5Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu -1 1 510 Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile 15 2025 30 Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp 3540 45 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr 5055 60 Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly 6570 75 Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg 8085 90 Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr 95100 105 110 Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg ArgIle 115 120 125 Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu SerTrp Leu 130 135 140 Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr ValSer Gln Asp 145 150 155 Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys LeuAsp Phe Asn Met 160 165 170 Thr Thr Asn His Ser Phe Met Cys Leu Ile LysTyr Gly His Leu Arg 175 180 185 190 Val Asn Gln Thr Phe Asn Trp Asn ThrThr Lys Gln Glu His Phe Pro 195 200 205 Asp Asn Leu Leu Pro Ser Trp AlaIle Thr Leu Ile Ser Val Asn Gly 210 215 220 Ile Phe Val Ile Cys Cys LeuThr Tyr Cys Phe Ala Pro Arg Cys Arg 225 230 235 Glu Arg Arg Arg Asn GluArg Leu Arg Arg Glu Ser Val Arg Pro Val 240 245 250 (2) INFORMATION FORSEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1716 base pairs(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL: no (vi) ORIGINALSOURCE: (A) ORGANISM: Mus musculus (D) DEVELOPMENTAL STAGE: germ line(F) TISSUE TYPE: lymphoid (G) CELL TYPE: B lymphocyte (H) CELL LINE: 70Zand A20 (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA in pCDM8 vector (B)CLONE: B7 #′s 1 and 29 (ix) FEATURE: (A) NAME/KEY: translated region (B)LOCATION: 249 to 1166 bp (C) IDENTIFICATION METHOD: similarity to otherpattern (ix) FEATURE: (A) NAME/KEY: Alternate ATG initiation codons (B)LOCATION: 225 to 227 and 270 to 272 (C) IDENTIFICATION METHOD:similarity to other pattern (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:GAGTTTTATA CCTCAATAGA CTCTTACTAG TTTCTCTTTT TCAGGTTGTG AAACTCAACC 60TTCAAAGACA CTCTGTTCCA TTTCTGTGGA CTAATAGGAT CATCTTTAGC ATCTGCCGGG 120TGGATGCCAT CCAGGCTTCT TTTTCTACAT CTCTGTTTCT CGATTTTTGT GAGCCTAGGA 180GGTGCCTAAG CTCCATTGGC TCTAGATTCC TGGCTTTCCC CATCATGTTC TCCAAAGCAT 240CTGAAGCT ATG GCT TGC AAT TGT CAG TTG ATG CAG GAT ACA CCA CTC CTC 290 MetAla Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu -35 -30 -25 AAG TTTCCA TGT CCA AGG CTC AAT CTT CTC TTT GTG CTG CTG ATT CGT 338 Lys Phe ProCys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg -20 -15 -10 CTT TCACAA GTG TCT TCA GAT GTT GAT GAA CAA CTG TCC AAG TCA GTG 386 Leu Ser GlnVal Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val -5 -1 1 5 AAA GATAAG GTA TTG CTG CCT TGC CGT TAC AAC TCT CCT CAT GAA GAT 434 Lys Asp LysVal Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp 10 15 20 25 GAG TCTGAA GAC CGA ATC TAC TGG CAA AAA CAT GAC AAA GTG GTG CTG 482 Glu Ser GluAsp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu 30 35 40 TCT GTC ATTGCT GGG AAA CTA AAA GTG TGG CCC GAG TAT AAG AAC CGG 530 Ser Val Ile AlaGly Lys Leu Lys Val Trp Pro Glu Tyr Lys Asn Arg 45 50 55 ACT TTA TAT GACAAC ACT ACC TAC TCT CTT ATC ATC CTG GGC CTG GTC 578 Thr Leu Tyr Asp AsnThr Thr Tyr Ser Leu Ile Ile Leu Gly Leu Val 60 65 70 CTT TCA GAC CGG GGCACA TAC AGC TGT GTC GTT CAA AAG AAG GAA AGA 626 Leu Ser Asp Arg Gly ThrTyr Ser Cys Val Val Gln Lys Lys Glu Arg 75 80 85 GGA ACG TAT GAA GTT AAACAC TTG GCT TTA GTA AAG TTG TCC ATC AAA 674 Gly Thr Tyr Glu Val Lys HisLeu Ala Leu Val Lys Leu Ser Ile Lys 90 95 100 105 GCT GAC TTC TCT ACCCCC AAC ATA ACT GAG TCT GGA AAC CCA TCT GCA 722 Ala Asp Phe Ser Thr ProAsn Ile Thr Glu Ser Gly Asn Pro Ser Ala 110 115 120 GAC ACT AAA AGG ATTACC TGC TTT GCT TCC GGG GGT TTC CCA AAG CCT 770 Asp Thr Lys Arg Ile ThrCys Phe Ala Ser Gly Gly Phe Pro Lys Pro 125 130 135 CGC TTC TCT TGG TTGGAA AAT GGA AGA GAA TTA CCT GGC ATC AAT ACG 818 Arg Phe Ser Trp Leu GluAsn Gly Arg Glu Leu Pro Gly Ile Asn Thr 140 145 150 ACA ATT TCC CAG GATCCT GAA TCT GAA TTG TAC ACC ATT AGT AGC CAA 866 Thr Ile Ser Gln Asp ProGlu Ser Glu Leu Tyr Thr Ile Ser Ser Gln 155 160 165 CTA GAT TTC AAT ACGACT CGC AAC CAC ACC ATT AAG TGT CTC ATT AAA 914 Leu Asp Phe Asn Thr ThrArg Asn His Thr Ile Lys Cys Leu Ile Lys 170 175 180 185 TAT GGA GAT GCTCAC GTG TCA GAG GAC TTC ACC TGG GAA AAA CCC CCA 962 Tyr Gly Asp Ala HisVal Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro 190 195 200 GAA GAC CCT CCTGAT AGC AAG AAC ACA CTT GTG CTC TTT GGG GCA GGA 1010 Glu Asp Pro Pro AspSer Lys Asn Thr Leu Val Leu Phe Gly Ala Gly 205 210 215 TTC GGC GCA GTAATA ACA GTC GTC GTC ATC GTT GTC ATC ATC AAA TGC 1058 Phe Gly Ala Val IleThr Val Val Val Ile Val Val Ile Ile Lys Cys 220 225 230 TTC TGT AAG CACAGA AGC TGT TTC AGA AGA AAT GAG GCA AGC AGA GAA 1106 Phe Cys Lys His ArgSer Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu 235 240 245 ACA AAC AAC AGCCTT ACC TTC GGG CCT GAA GAA GCA TTA GCT GAA CAG 1154 Thr Asn Asn Ser LeuThr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln 250 255 260 265 ACC GTC TTCCTT TAGTTCTTCT CTGTCCATGT GGGATACATG GTATTATGTG 1206 Thr Val Phe LeuGCTCATGAGG TACAATCTTT CTTTCAGCAC CGTGCTAGCT GATCTTTCGG ACAACTTGAC 1266ACAAGATAGA GTTAACTGGG AAGAGAAAGC CTTGAATGAG GATTTCTTTC CATCAGGAAG 1326CTACGGGCAA GTTTGCTGGG CCTTTGATTG CTTGATGACT GAAGTGGAAA GGCTGAGCCC 1386ACTGTGGGTG GTGCTAGCCC TGGGCAGGGG CAGGTGACCC TGGGTGGTAT AAGAAAAAGA 1446GCTGTCACTA AAAGGAGAGG TGCCTAGTCT TACTGCAACT TGATATGTCA TGTTTGGTTG 1506GTGTCTGTGG GAGGCCTGCC CTTTTCTGAA GAGAAGTGGT GGGAGAGTGG ATGGGGTGGG 1566GGCAGAGGAA AAGTGGGGGA GAGGGCCTGG GAGGAGAGGA GGGAGGGGGA CGGGGTGGGG 1626GTGGGGAAAA CTATGGTTGG GATGTAAAAA CGGATAATAA TATAAATATT AAATAAAAAG 1686AGAGTATTGA GCAAAAAAAA AAAAAAAAAA 1716 (2) INFORMATION FOR SEQ ID NO:31:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 306 amino acids (B) TYPE:amino acid (C) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (A)DESCRIPTION: B lymphocyte activation antigen; Ig superfamily member; Tcell costimulatory signal via activation of CD28 pathways, binds toCD28+ T cells, transmembrane protein (ix) FEATURE: (A) NAME/KEY: signalsequence (B) LOCATION: -37 to -1 (C) IDENTIFICATION METHOD: similaritywith known sequence (D) OTHER INFORMATION: hydrophobic (ix) FEATURE: (A)NAME/KEY: extracellular domain (B) LOCATION: 1 to 210 (C) IDENTIFICATIONMETHOD: similarity with known sequence (ix) FEATURE: (A) NAME/KEY:transmembrane domain (B) LOCATION: 211 to 235 (C) IDENTIFICATION METHOD:similarity with known sequence (ix) FEATURE: (A) NAME/KEY: intracellular(cytoplasmic) domain (B) LOCATION: 236 to 269 (C) IDENTIFICATION METHOD:similarity with known sequence (ix) FEATURE: (A) NAME/KEY: Ig V-setdomain (B) LOCATION: 1 to 105 (C) IDENTIFICATION METHOD: similarity withknown sequence (ix) FEATURE: (A) NAME/KEY: Ig C-set domain (B) LOCATION:106 to 199 (C) IDENTIFICATION METHOD: similarity with known sequence (x)PUBLICATION INFORMATION: (A) AUTHORS: FREEMAN, GORDON J. GRAY, GARY S.GIMMI, CLAUDE D. LOMBARD, DAVID B. ZHOU, LIANG-JI WHITE, MICHAELFINGEROTH, JOYCE D. GRIBBEN, JOHN G. NADLER, LEE M. (B) TITLE:Structure, Expression, and T Cell Costimulatory Activity Of The MurineHomologue Of The Human B Lymphocyte Activation Antigen B7 (C) JOURNAL:Journal of Experimental Medicine (D) VOLUME: (E) ISSUE: (F) PAGES: (G)DATE: IN PRESS (H) RELEVANT RESIDUES IN SEQ ID NO:31: From -37 to 269(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Met Ala Cys Asn Cys Gln Leu MetGln Asp Thr Pro Leu Leu Lys Phe -35 -30 -25 Pro Cys Pro Arg Leu Ile LeuLeu Phe Val Leu Leu Ile Arg Leu Ser -20 -15 -10 Gln Val Ser Ser Asp ValAsp Glu Gln Leu Ser Lys Ser Val Lys Asp -5 -1 1 5 10 Lys Val Leu Leu ProCys Arg Tyr Asn Ser Pro His Glu Asp Glu Ser 15 20 25 Glu Asp Arg Ile TyrTrp Gln Lys His Asp Lys Val Val Leu Ser Val 30 35 40 Ile Ala Gly Lys LeuLys Val Trp Pro Glu Tyr Lys Asn Arg Thr Leu 45 50 55 Tyr Asp Asn Thr ThrTyr Ser Leu Ile Ile Leu Gly Leu Val Leu Ser 60 65 70 75 Asp Arg Gly ThrTyr Ser Cys Val Val Gln Lys Lys Glu Arg Gly Thr 80 85 90 Tyr Gly Val LysHis Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp 95 100 105 Phe Ser ThrPro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr 110 115 120 Lys ArgIle Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe 125 130 135 SerTrp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile 140 145 150155 Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln Leu Asp 160165 170 Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly175 180 185 Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro GluAsp 190 195 200 Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala GlyPhe Gly 205 210 215 Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile LysCys Phe Cys 220 225 230 235 Lys His Arg Ser Cys Phe Arg Arg Asn Glu AlaSer Arg Glu Thr Asn 240 245 250 Asn Ser Leu Thr Phe Gly Pro Glu Glu AlaLeu Ala Glu Gln Thr Val 255 260 265 Phe Leu (2) INFORMATION FOR SEQ IDNO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:GGCACTAGGT CTCCAGCTTG AGATCACAGT TCTCTCTAC 39 (2) INFORMATION FOR SEQ IDNO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:GCTTGAATCT TCAGAGGAGC GGAGTGGACA CCTGTGG 37 (2) INFORMATION FOR SEQ IDNO:34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:GCTCCTCTGA AGATTCAAGC 20 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:35: GGCACTATGA TCAGGGGGAG GCTGAGGTCC 30(2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 78 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:36: GCATTTTAAG CTTTTTCCTG ATCAGGAGCC CAAATCTTCTGACAAAACTC ACACATCTCC 60 ACCGTCTCCA GGTAAGCC 78 (2) INFORMATION FOR SEQID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:TAATACGACT CACTATAGGG 20 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:38: GAGCATTTTC CTGATCAGGA GTCCAAATATGGTCCCCCAT CCCATCATCC CCAGGTAAGC 60 CAACCC 66 (2) INFORMATION FOR SEQ IDNO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GCAGAGGAATCGAGCTCGGT ACCCGGGGAT CCCCAGTGTG GGGACAGTGG GACCGCTCTG 60 CCTCCC 66 (2)INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:59 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQID NO:40: GGGTTTTGGG GGGAAGAGGA AGACTGACGG TGCCCCCTCG GCTTCAGGTGCTGAGGAAG 59 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi)SEQUENCE DESCRIPTION: SEQ ID NO:41: CATCTCTTCC TCAGCACCTG AAGCCGAGGGGGCACCGTCA GTCTTCCTCT TCCCCC 56 (2) INFORMATION FOR SEQ ID NO:42: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 99 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CGCACGTGACCTCAGGGGTC CGGGAGATCA TGAGAGTGTC CTTGGGTTTT GGGGGGAACA 60 GGAAGACTGATGGTGCCCCC TCGAACTCAG GTGCTGAGG 99 (2) INFORMATION FOR SEQ ID NO:43: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 98 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: CCTCAGCACCTGAGTTCGAG GGGGCACCAT CAGTCTCCTG TTCCCCCCAA AACCCAAGGA 60 CACTCTCATGATCTCCCGGA CCCCTGAGGT CACGTGCG 98 (2) INFORMATION FOR SEQ ID NO:44: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 330 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..330 (xi) SEQUENCEDESCRIPTION: SEQ ID NO:44: GCT CCT CTG AAG ATT CAA GCT TAT TTC AAT GAGACT GCA GAC CTG CCA 48 Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu ThrAla Asp Leu Pro 1 5 10 15 TGC CAA TTT GCA AAC TCT CAA AAC CAA AGC CTGAGT GAG CTA GTA GTA 96 Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu SerGlu Leu Val Val 20 25 30 TTT TGG CAG GAC CAG GAA AAC TTG GTT CTG AAT GAGGTA TAC TTA GGC 144 Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu ValTyr Leu Gly 35 40 45 AAA GAG AAA TTT GAC AGT GTT CAT TCC AAG TAT ATG GGCCGC ACA AGT 192 Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly ArgThr Ser 50 55 60 TTT GAT TCG GAC AGT TGG ACC CTG AGA CTT CAC AAT CTT CAGATC AAG 240 Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln IleLys 65 70 75 80 GAC AAG GGC TTG TAT CAA TGT ATC ATC CAT CAC AAA AAG CCCACA GGA 288 Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro ThrGly 85 90 95 ATG ATT CGC ATC CAC CAG ATG AAT TCT AGG CTG TCA GTG CTT 330Met Ile Arg Ile His Gln Met Asn Ser Arg Leu Ser Val Leu 100 105 110 (2)INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:110 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: Ala Pro Leu LysIle Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu Pro 1 5 10 15 Cys Gln PheAla Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val Val 20 25 30 Phe Trp GlnAsp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu Gly 35 40 45 Lys Glu LysPhe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr Ser 50 55 60 Phe Asp SerAsp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile Lys 65 70 75 80 Asp LysGly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr Gly 85 90 95 Met IleArg Ile His Gln Met Asn Ser Arg Leu Ser Val Leu 100 105 110 (2)INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:306 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY:CDS (B) LOCATION: 1..310 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GCTAAC TTC AGT CAA CCT GAA ATA GTA CCA ATT TCT AAT ATA ACA GAA 48 Ala AsnPhe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu 1 5 10 15 AATGTG TAC ATA AAT TTG ACC TGC TCA TCT ATA CAC GGT TAC CCA GAA 96 Asn ValTyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu 20 25 30 CCT AAGAAG ATG AGT GTT TTG CTA AGA ACC AAG AAT TCA ACT ATC GAG 144 Pro Lys LysMet Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu 35 40 45 TAT GAT GGTATT ATG CAG AAA TCT CAA GAT AAT GTC ACA GAA CTG TAC 192 Tyr Asp Gly IleMet Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr 50 55 60 GAC GTT TCC ATCAGC TTG TCT GTT TCA TTC CCT GAT GTT ACG AGC AAT 240 Asp Val Ser Ile SerLeu Ser Val Ser Phe Pro Asp Val Thr Ser Asn 65 70 75 80 ATG ACC ATC TTCTGT ATT CTG GAA ACT GAC AAG ACG CGG CTT TTA TCT 288 Met Thr Ile Phe CysIle Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser 85 90 95 TCA CCT TTC TCT ATAGAG 306 Ser Pro Phe Ser Ile Glu 100 (2) INFORMATION FOR SEQ ID NO:47:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 102 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO:47: Ala Asn Phe Ser Gln Pro Glu Ile ValPro Ile Ser Asn Ile Thr Glu 1 5 10 15 Asn Val Tyr Ile Asn Leu Thr CysSer Ser Ile His Gly Tyr Pro Glu 20 25 30 Pro Lys Lys Met Ser Val Leu LeuArg Thr Lys Asn Ser Thr Ile Glu 35 40 45 Tyr Asp Gly Ile Met Gln Lys SerGln Asp Asn Val Thr Glu Leu Tyr 50 55 60 Asp Val Ser Ile Ser Leu Ser ValSer Phe Pro Asp Val Thr Ser Asn 65 70 75 80 Met Thr Ile Phe Cys Ile LeuGlu Thr Asp Lys Thr Arg Leu Leu Ser 85 90 95 Ser Pro Phe Ser Ile Glu 100(2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:48: GCAACCGGAA GCTTGCCACC ATGGGGGTAC TGCTCACACAGAGGACG 47 (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 63 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: AGTCTCATTGAAATAAGCTT GAATCTTCAG AGGAGCCATG CTGGCCATGC TTGGAAACAG 60 GAG 63 (2)INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:50: CTCCTGTTTC CAAGCATGGC CAGCATGGCT CCTCTGAAGATTCAGGCTTA TTTCAATGAG 60 AC 62 (2) INFORMATION FOR SEQ ID NO:51: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: TGTGTGTGGAATTCTCATTA CTGATCAAGC ACTGACAGTT CAGAATTCAT C 51 (2) INFORMATION FOR SEQID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 63 base pairs (B)TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)MOLECULE TYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:AGAAATTGGT ACTATTTCAG GTTGACTGAA GTTAGCCATG CTGGCCATGC TTGGAAACAG 60 GAG63 (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 62 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO:53: CTCCTGTTTC CAAGCATGGC CAGCATGGCT AACTTCAGTCAACCTGAAAT AGTACCAATT 60 TC 62 (2) INFORMATION FOR SEQ ID NO:54: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 105 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CTCAAGCTTGCCACCATGGG GGTACTGCTC ACACAGAGGA CGCTGCTCAG TCTGGTCCTT 60 GCACTCCTGTTTCCGAGCAT GGCGAGCATG GGTCTTTCTC ACTTC 105 (2) INFORMATION FOR SEQ IDNO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:TGTGTGTGGA ATTCTCATTA CTGATCAGGA AAATGCTCTT GCTTG 45

1. An isolated nucleic acid encoding a B7-2 fusion protein comprising anucleotide sequence encoding a first peptide having a B7-2 activity anda nucleotide sequence encoding a second peptide corresponding to amoiety that alters the solubility, binding affinity or valency of thefirst peptide.
 2. The isolated nucleic acid of claim 1 which is a DNA.3. The isolated nucleic acid of claim 2, wherein the first peptidecomprises an extracellular domain of a human B7-2 protein.
 4. Theisolated nucleic acid of claim 3, wherein the first peptide comprisesamino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ IDNO:2).
 5. The isolated nucleic acid of claim 3, wherein the firstpeptide comproses a variable region-like domain of human B7-2.
 6. Theisolated nucleic acid of claim 3, wherein the first peptide comprises aconstant region-like domain of human B7-2.
 7. The isolated nucleic acidof claim 2, wherein the second peptide comprises an immonoglobulinconstant region.
 8. The isolated nucleic acid of claim 7, wherein theimmunoglobulin constant region is a Cγ1 domain, including the hinge, CH2and CH3 region.
 9. The isolated nucleic acid of claim 7, wherein theimmunoglobulin constant region is modified to reduce constantregion-mediated biological effector functions.
 10. The isolated nucleicacid of claim 9, wherein the biological effector function is selectedfrom the group consisting of complement activation, Fc receptorinteraction, and complement activation and Fc receptor interaction. 11.The isolated nucleic acid of claim 10, wherein the immunoglobulinconstant region is a Cγ4 domain, including the hinge, CH2 and CH3region.
 12. The isolated nucleic acid of claim 11, wherein at least oneamino acid residue of the CH2 domain is modified by substitution,addition or deletion.
 13. An isolated B7-2 fusion protein comprising afirst peptide having a B7-2 activity and a second peptide correspondingto a moiety that alters the solubility, binding affinity or valency ofthe first peptide.
 14. The isolated B7-2 fusion protein of claim 13,wherein the first peptide comprises an extracellular domain of humanB7-2 protein.
 15. The isolated B7-2 fusion protein of claim 14, whereinthe first peptide comprises amino acid residues 24-245 of the sequenceshown in FIG. 8 (SEQ ID NO:2).
 16. The isolated B7-2 fusion protein ofclaim 14, wherein the first peptide comprises a variable region-likedomain of human B7-2.
 17. The isolated B7-2 fusion protein of claim 14,wherein the first peptide comprises a constant region-like domain ofhuman B7-2.
 18. The isolated B7-2 fusion protein of claim 13, whereinthe second peptide comprises an immonoglobulin constant region.
 19. Theisolated B7-2 fusion protein of claim 18, wherein the immunoglobulinconstant region is a Cγ1 domain, including the hinge, CH2 and CH3region.
 20. The isolated B7-2 fusion protein of claim 18, wherein theimmunoglobulin constant region is modified to reduce constantregion-mediated biological effector functions.
 21. The isolated B7-2fusion protein of claim 20, wherein the biological effector function isselected from the group consisting of complement activation, Fc receptorinteraction, and complement activation and Fc receptor interaction. 22.The isolated B7-2 fusion protein of claim 21, wherein the immunoglobulinconstant region is a Cγ4 domain, including the hinge, CH2 and CH3region.
 23. The isolated B7-2 fusion protein of claim 22, wherein atleast one amino acid residue of the CH2 domain is modified bysubstitution, addition or deletion.
 24. A composition suitable forpharmaceutical administration comprising a fusion protein of claim 13and a pharmaceutically acceptable carrier.
 25. A composition suitablefor pharmaceutical administration comprising a fusion protein of claim14 and a pharmaceutically acceptable carrier.
 26. A composition suitablefor pharmaceutical administration comprising a fusion protein of claim16 and a pharmaceutically acceptable carrier.
 27. A composition suitablefor pharmaceutical administration comprising a fusion protein of claim18 and a pharmaceutically acceptable carrier.
 28. A method forinhibiting an interaction of a B lymphocyte antigen, B7-2, with itsnatural ligand(s) on the surface of immune cells, comprising contactingan immune cell with a B7-2 fusion protein which inhibits B7-2 bindingwith its natural ligand(s), to thereby inhibit costimulation of theimmune cell through the B7-2-ligand interaction.
 29. The method of claim28, wherein the B7-2 fusion protein comprises a first peptide havingB7-2 activity and a second peptide comprising a moiety that alters thesolubility, binding affinity or valency of the first peptide.
 30. Themethod of claim 29, wherein the first peptide comprises an extracellulardomain of the human B7-2 protein.
 31. The method of claim 30, whereinthe first peptide comprises amino acid residues 24-245 of the sequenceshown in FIG. 8 (SEQ ID NO:2).
 32. The method of claim 29, wherein thesecond peptide comprises an immonoglobulin constant region.
 33. Themethod of claim 32, wherein the immunoglobulin constant region is a Cγ1domain, including the hinge, CH2 and CH3 region.
 34. A method fortreating an autoimmune disease in a subject mediated by interaction of aB lymphocyte antigen, B7-2, with its natural ligand(s) on the surface ofimmune cells, comprising administering to the subject an inhibitory formof a B7-2 fusion protein, to thereby inhibit costimulation of the immunecells through the B7-2-ligand interaction.
 35. The method of claim 34,wherein the inhibitory form of a B7-2 fusion protein is a B7-2immunoglobulin fusion protein (B7-2Ig) comprising a first peptidecomprising an extracellular domain of the B7-2 protein and a secondpeptide comprising an immunoglobulin constant domain.
 36. The method ofclaim 35, wherein the extracellular domain of the B7-2 protein comprisesamino acid residues 24-245 of the sequence shown in FIG. 8 (SEQ IDNO:2).
 37. A method for treating allergy in a subject mediated byinteraction of a B lymphocyte antigen, B7-2, with its natural ligand(s)on the surface of immune cells, comprising administering to the subjectan inhibitory form of a B7-2 fusion protein, to thereby inhibitcostimulation of the immune cells through the B7-2-ligand interaction.38. An isolated variable region form of the B cell activation antigenB7-2 which comprises a B7-2 immunoglobulin-like variable region domainbut does not comprise a B7-2 immunoglobulin-like constant region domain.39. The B7-2 variable region form of claim 38, which is human.
 40. TheB17-2 variable region form of claim 38, which is a fusion proteincomprising a B7-2 variable region polypeptide operatively linked to aheterologous polypeptide.
 41. The B7-2 variable region form of claim 40,wherein the B7-2 variable region polypeptide is a human B7-2 variableregion polypeptide.
 42. The B7-2 variable region form of claim 41,wherein the human B7-2 variable region polypeptide comprises an aminoacid sequence of about positions 24 to 133 of SEQ ID NO:
 2. 43. The B7-2variable region form of claim 40, wherein the heterologous polypeptidecomprises an immunoglobulin constant region.
 44. The B7-2 variableregion form of claim 43, wherein the immunoglobulin constant regioncomprises the hinge, CH2 and CH3 domains of IgG1.
 45. The B7-2 variableregion form of claim 38, comprising a B7-2 immunoglobulin-like variableregion domain operatively linked to a transmembrane domain, the B7-2variable region form being expressed on the surface of a cell.
 46. TheB7-2 variable region form of claim 45, further comprising a non-B7-2linker polypeptide located between the B7-2 immunoglobulin-like variableregion domain and the transmembrane domain.
 47. The B7-2 variable regionform of claim 45, further comprising a cytoplasmic domain.
 48. The B7-2variable region form of claim 38, comprising a B7-2 immunoglobulin-likevariable region domain bound to a solid support.
 49. The B7-2 variableregion form of claim 48, wherein the solid support is a bead or plate.50. The B7-2 variable region form of claim 48, further comprising anon-B7-2 linker polypeptide located between the B7-2 immunoglobulin-likevariable region domain and the solid support.
 51. An isolated B7-2fusion protein comprising a human B7-2 immunoglobulin-like variableregion domain operatively linked to a heterologous polypeptide, whereinthe B7-2 fusion protein does not comprise a B7-2 immunoglobulin-likeconstant region domain.
 52. The B7-2 fusion protein of claim 51, whereinthe human B7-2 immunoglobulin-like variable region domain comprises anamino acid sequence from about position 24 to position 133 of SEQ ID NO:2.
 53. The B7-2 fusion protein of claim 51, wherein the heterologouspolypeptide comprises an immunoglobulin constant region polypeptide. 54.An isolated nucleic acid molecule encoding a variable region form of aB7-2 fusion protein, the B7-2 fusion protein comprising a human B7-2immunoglobulin-like variable region domain operatively linked to aheterologous polypeptide, wherein the B7-2 fusion protein does notcomprise a B7-2 immunoglobulin-like constant region domain.
 55. Thenucleic acid of claim 58, wherein the heterologous polypeptide is animmunoglobulin constant region polypeptide.
 56. A recombinant expressionvector comprising the nucleic acid molecule of claim
 54. 57. A host cellcontaining the recombinant expression vector of claim
 56. 58. Anisolated nucleic acid molecule encoding a variable region form of B7-2,the nucleic acid comprising a contiguous nucleotide sequence encoding asignal peptide, a human B7-2 immunoglobulin-like variable region domain,a transmembrane domain and a cytoplasmic domain.
 59. The nucleic acidmolecule of claim 58, wherein the human B7-2 immunoglobulin-likevariable region domain comprises an amino acid sequence from aboutposition 24 to position 133 of SEQ ID NO:
 2. 60. The nucleic acidmolecule of claim 58, further comprising a nucleotide sequence encodinga non-B7-2 linker polypeptide located between the nucleotide sequenceencoding the B7-2 immunoglobulin-like variable region domain and thetransmembrane domain.
 61. A recombinant expression vector comprising thenucleic acid molecule of claim
 58. 62. A host cell containing therecombinant expression vector of claim 61, wherein the variable regionform of B7-2 is expressed on the surface of the cell.
 63. A method forstimulating a response by an activated T cell, comprising contacting theactivated T cell with a variable region form of the B cell activationantigen B7-2, the variable region form of B7-2 comprising a B7-2immunoglobulin-like variable region domain but not comprising a B7-2immunoglobulin-like constant region domain such that a response by theactivated T cell is stimulated.
 64. The method of claim 63, wherein aT_(helper)-Type 2 (TH₂) response is preferentially stimulated.